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DD34E DNA Transposable Elements of Mosquitoes: Whole-genome survey, evolution, and transposition
Monique R. Coy
Dissertation submitted to the faculty of the Virginia Polytechnic Institute and State University in partial fulfillment of the requirements for the degree of
Doctor of Philosophy
In Biochemistry
Zhijian (Jake) Tu, Chair Zachary Adelman Glenda Gillaspy Peter J Kennelly John McDowell
(13th of June 2007) Blacksburg, Virginia
Keywords: DNA transposable element, evolution, horizontal transfer, gambol, interspersed repeat, mosquito, Tc1, transposition assay
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DD34E DNA Transposable Elements of Mosquitoes: Whole-genome survey, evolution,
and transposition
Monique Royer Coy
Abstract
ii
Transposable elements (TEs) are mobile genetic elements capable of replicating and
spreading within, and in some cases, between genomes. I describe a whole-genome analysis
of DD34E TEs, which belong to the IS630-Tc1-mariner superfamily of DNA transposable
elements, in the African malaria mosquito, Anopheles gambiae. Twenty-six new transposons
as well as a new family, gambol, were identified. The gambol family shares the DD34E
catalytic motif with Tc1-DD34E transposons, but is distinct from these elements in their
phylogenetic relationships. Although gambol appears to be related to a few DD34E
transposons from cyanobacteria and fungi, no gambol elements have been reported in any
other insects or animals thus far. This discovery expands the already expansive diversity of
the IS630-Tc1-mariner TEs, and raises interesting questions as to the origin of gambol
elements and their apparent diversity in An. gambiae. Several DD34E transposons discovered
in An. gambiae possess characteristics that are associated with recent transposition, such as
high sequence identity between copies, and intact terminal-inverted repeats and open reading
frames. One such element, AgTango, was also found in a distantly related mosquito species,
Aedes aegypti, at high amino acid sequence identity (79.9%). It was discovered that Tango
transposons have patchy distribution among twelve mosquito species surveyed using PCR as
well as genomic searches, suggesting a possible case for horizontal transfer. Additionally, it
was discovered that in some mosquito genomes, there are several Tango transposons. These
observations suggest differential evolutionary scenarios and/or TE-host interaction of Tango
elements between mosquito species. This strengthened the case that AgTango may be a
functional transposase, and I sought to test its potential activity in a cell culture-based inter-
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plasmid transposition assay using the Herves plasmids as a positive control (Arensburger et
al., 2005). AgTango constructs were successfully constructed; however, no transposition
events were detected for Tango or Herves. Because the positive control failed to work, no
assessment can be made concerning Tango’s transposase. Possible causes and solutions for
these results, alternative means to detect transposition, as well as future directions with Tango
are discussed.
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Acknowledgements
To Jake, my dissertation advisor:
Thank you for providing me with a nurturing, intellectual environment and giving me
freedom and control over my project. You have many qualities that make you an excellent
advisor, but most of all I appreciated your security and patience to allow me to make
mistakes.
To Dr. Peter Kennelly:
I was fortunate to have not one advisor, but two. I appreciate the dedication you have shown
towards my education and development as a scientist. Thank you very much for the times that
you were my sounding board and gave me a sense of balance as I worked my way through
my graduate education.
To my committee members – past and present:
Thank you for your dedication and time to my education. From the classes I took with you to
my committee meetings, it was apparent that you cared about my education. Something I
learned early on in my graduate career was that professors have many obligations. Yet, every
time we met, it was obvious to me that you had read the materials that I provided and
considered carefully them before we met. I realize now how difficult that truly is, and I
appreciate it very much.
To Thomas E Coy, my husband:
Tom, this was a joint effort and I could have never accomplished it without you. This Ph.D. is
as much yours as it is mine. I appreciate your efforts to understand my ramblings, and your
tolerance of my piles of journal articles that litter our home, and the dead bugs in our
refrigerator. Never in my wildest dreams did I ever imagine that I would find somebody who
would be able to understand and accept my quirkiness, much less love and encourage it.
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Most importantly, thank you for cultivating my sense of humor as this would have been
impossible otherwise.
To Nicholas D Coy, my son:
Never before have I wanted to get my act together so badly until the day I looked into your
eyes for the first time. Your arrival gave me focus and a renewed point to my life. I hope you
will never tire of me telling you about the physical laws of nature, teaching you birdcalls, and
hugging you.
To David L Royer, my dad and Mona Royer, my mom:
Dad, thank you for grounding me early in the concept that the world is a physical place that
obeys laws which can be studied and understood. You taught me that things are knowable,
understandable, often predictable, and to a certain degree, controllable. Because of this, I
have avoided the pitfall of depending upon superstition to understand the world, which is the
greatest gift you could have given me. Mom, thank you for hanging a butterfly net on my
wall as a decoration, and didn’t kill me later when I used it for its purpose to let loose
butterflies in my bedroom and to show you how they roll their tongues in and out. I
appreciate your “tolerance” of the Petri dishes under my bed. And, although I’ll never admit
it in person, thank you for dragging me all over the world to visit art museums. To both of
you, thank you both for never, ever, discouraging or limiting me from doing things because I
was a “girl”.
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Attribution
Work presented in Chapters 2 and 3 are reprinted from the journal Insect Molecular Biology.
My chair and primary advisor, Dr. Zhijian (Jake) Tu, is a coauthor on both publications. He
provided guidance of the research presented in these chapters, and edited and revised the
drafts prepared for publication. Dr. Tu obtained his PhD in entomology at the University of
Arizona. He is currently an associate professor in the biochemistry department at Virginia
Tech.
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Table of Contents
Abstract ii
Acknowledgements iv
Attribution vi
Table of Contents vii
List of Figures x
List of Tables xii
List of Abbreviations xiii
Chapter 1 Introduction 1
1.1 Transposable Elements
1.1.1 Transposable Elements Defined 1
1.1.2 Classification of Transposable elements 2
1.1.3 IS630-Tc1-mariner Superfamily of Class II Transposable Elements 3
1.1.4 Evolutionary Impact of Transposable Elements on Genome Function
and Evolution 5
1.1.5 Horizontal Transfer of Transposable Elements 8
1.1.6 Utility of Transposable Elements as Biological Tools 9
1.1.7 Identification of Active Elements 10
1.2 Mosquitoes
1.2.1 Anopheles gambiae 11
1.2.2 Mosquito Taxonomy and Taxonomic Relationships 12
1.2.3 Controlling Disease through Genetic Manipulation 14
1.3 Dissertation Scheme
1.3.1 Research Aims and Scheme 16
Chapter 2 Gambol and Tc1 are two distinct families of DD34E transposons:
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Analysis of the Anopheles gambiae genome expands the diversity of
the IS630-Tc1-mariner superfamily 17
2.1 Abstract 17
2.2 Introduction 18
2.3 Materials and Methods 21
2.4 Results 25
2.5 Discussion 30
2.6 Acknowledgements 33
Chapter 3 Genomic and evolutionary analyses of Tango transposons in
Aedes aegypti, Anopheles gambiae, and other mosquito species 44
3.1 Abstract 44
3.2 Introduction 45
3.3 Materials and Methods 47
3.4 Results 53
3.5 Discussion 58
3.6 Acknowledgements 62
Chapter 4 Inter-plasmid transposition assay to test the functionality
of the AgTango transposase 77
4.1 Abstract 77
4.2 Introduction 78
4.3 Materials and Methods 80
4.4 Results 89
4.5 Discussion 92
4.6 Acknowledgements 98
References 111
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Appendix A Identities at the amino acid levels of different gene products and
Tango between Anopheles gambiae and Aedes aegypti 129
Curriculum vitae 130
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List of Figures
Figure 2.1 Select sections of the multiple sequence alignment of the An. gambiae
DD34E TEs and IS630-Tc1-mariner superfamily representatives 35
Figure 2.2 The DNA binding domains of Tc3 and Sleeping Beauty (SB) compared
to the predicted HTH motifs of the gambol elements and two mariner
TEs with which the gambol elements share a similar N-terminal
signature (XXDEDC) 37
Figure 2.3 Phylogenetic relationships between An. gambiae DD34E and
representative IS630-Tc1-mariner superfamily transposases 38
Figure 2.4 TIR consensus sequences in WebLogo format for the first 24 nucleotides
of the gambol and Tc1 TEs from An. gambiae, and ITmDD37D TEs
from Caenorhabditis elegans, C. briggsae, and An. gambiae 40
Figure 2.5 Density of gambol and Tc1 TEs per chromosomal arm of the
An. gambiae genome 41
Figure 3.1 BLASTp alignment showing high sequence similarity between
AgTango1 and AeTango1 64
Figure 3.2 Inferred phylogenetic relationships between Aedes aegypti Tango
elements, AgTango1 from Anopheles gambiae, and representative
Tc1 from Ae. aegypti and other organisms 66
Figure 3.3 Conserved features and sequences of the terminal inverted repeats
(TIRs) of Tango transposons 68
Figure 3.4 Inferred phylogenetic relationship of Tango elements 70
Figure 3.5 Comparison of selection pressure on Tango1 and four host
genes of Anopheles gambiae and Aedes aegypti 73
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Figure 4.1 Schematic of AgTango, a Tc1 transposable element from Anopheles
gambiae 99
Figure 4.2 Maps of pBSHvSacOα (Donor Plasmid) and pBSHvOα
(Donor Plasmid – Revised) 101
Figure 4.3 A reverse-contrast photograph of an ethidium bromide-stained
1% agarose-TAE gel showing the results of PstI restriction digest of
pBSHvSacOα (Donor Plasmid) 104
Figure 4.3 Amino acid translations of the PCR sequence of AgTango’s ORF
(AgTangoAmp) vs. the sequence from the Anopheles gambiae genome
(AgTango) 105
Figure 4.5 Sequences from the 5’ and 3’ ends of a Herves recombinant
obtained in the interplasmid transposition assay 106
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List of Tables
Table 2.1 The DD34E transposable elements of Anopheles gambiae 42
Table 3.1 Percent amino acid identities between Tango transposons, three other
Tc1 elements from Aedes aegypti, and representative Tc1 elements
Quetzal and Sleeping Beauty 74
Table 3.2 The molecular characteristics and copy numbers of Tango transposons
from Aedes aegypti and Anopheles gambiae, and three other Tc1
elements from Ae. aegypti 75
Table 3.3. Distribution of Tango elements among mosquito species
surveyed using degenerate PCR 76
Table 4.1 Oligonucleotide primers used to sequence the ambiguous region
of pBSHvSacKOα 107
Table 4.2 Oligonucleotide primers to create Tango inserts for Tango Helper and
Donor plasmids for inter-plasmid transposition assay, and to sequence
recombinants to identify transposition reactions 108
Table 4.3 Oligonucleotide primers used to verify
Herves transposition events in products of interplasmid assay 109
Table 4.4 Results for inter-plasmid transposition assay for Herves and AgTango 110
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List of abbreviations
Amp ampicillin
bp base pair
Cam chloramphenicol
D aspartic acid
E glutamic acid
hsp heat-shock protein 70 coding region
Hsp heat-shock protein
HTH helix-turn-helix
IR/DR inverted repeat-direct repeat
IPTG isopropyl-β-D-thiogalactopyranoside
ITm IS630-Tc1-mariner
Kan kanamycin
lacZ β-galactosidase coding region from E. coli
LB Luria broth
uF micro-Faraday
MITE miniature inverted repeat transposable element
MYA million years ago
NLS nuclear localization signal
Ω Ohm
ORF open reading frame
ORI origin of replication
RT-PCR reverse transcriptase polymerase chain reaction
s.s. sensu stricto
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TE transposable element
TIR terminal inverted repeat
TSD target-site duplication
X-GAL 5-bromo-4-chloro-3-indolyl-β-D-galacto-pyranoside
V volts
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Chapter 1
INTRODUCTION
1.1 Transposable Elements
1.1.1 Transposable Elements Defined
Transposable elements (TEs) are genetic entities that move from one genomic location to
another, replicate and spread within a genome, and typically do not serve a function
directly to the genome in which they find themselves. In some cases, TEs are capable of
crossing species barriers to invade a new genome in a process called horizontal transfer.
TEs make up a significant portion of most eukaryotic genomes studied so far (Craig,
2002), and many TEs have been discovered and described. However, little is known
about their biological impact, evolution, or influence on their host genomes. TEs can
cause coding, regulatory and structural changes within the genome in a number of direct
and indirect ways. For example, upon integration, TEs can disrupt coding or regulatory
regions. Imprecise excision of a TE can result in portions of host DNA being removed
and relocated to a new genomic location, and footprints left behind can result in altered
gene products (Colot et al., 1998). Indirectly, TEs can facilitate genomic rearrangements
by providing regions of homology for ectopic recombination. Such changes can result in
new gene linkages.
With the explosion of available sequenced genomes for analysis, the significance
of TEs to genome evolution is beginning to be realized. In the past, TEs have been
regarded as “junk” (Doolittle & Sapienza, 1980) and “parasites” (Orgel & Crick, 1980),
but these viewpoints are beginning to be tempered. While it is agreed that in general TEs
are genomic parasites, using the cellular machinery of their hosts for their own
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propagation without providing a direct return, growing evidence suggests that these
elements can inadvertently play a positive role in genome evolution by providing
function and plasticity to their host genomes (reviewed in Kidwell & Lisch, 2001).
1.1.2 Classification of Transposable Elements
Transposable elements are classified as either Class I or Class II, depending on their
mode of transposition (Finnegan, 1992). The members of Class I transpose through an
RNA intermediate, and include the long terminal repeat retrotransposons (LTRs), non-
LTRs, and short interspersed nuclear elements (SINEs). Class II elements possess
terminal inverted repeats (TIRs) and transpose through a DNA intermediate, and include
DNA TEs such as Tc1 and hobo, miniature inverted repeat transposable elements
(MITEs), and helitrons. All members of this class transpose by a “cut-and-paste”
mechanism except for helitrons, which are believed to transpose by a rolling circle
mechanism e.g., (Plasterk et al., 1999).
Class II elements are further categorized based upon structural and mechanistic
characteristics, presumably reflecting evolutionary relationships. The terms superfamily,
family, subfamily, transposon, element, and copy are often used to describe relationships
of and between TEs, and in this dissertation will be used as follows: Going from
increasing degrees of relatedness, there are superfamilies, further broken down into
families and then into subfamilies, if applicable. IS630-Tc1-mariner and hAT are
examples of superfamilies. IS630, Tc1, and mariner are the founding families of the
IS630-Tc1-mariner superfamily (Plasterk et al., 1999; Shao & Tu, 2001). The members
of a family are a group of related transposons found in diverse organisms, and usually
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share conserved amino acid sequences in their transposase. For example, the TEs Impala,
Sleeping Beauty and Topi are transposons of the Tc1 family from fungus, fish, and
mosquito, respectively (Langin et al., 1995; Ivics et al., 1997; Grossman et al., 1999).
Likewise, multiple transposons from a given family can occupy a genome. For example,
Topi, Tiang and Tsessebe are Tc1 transposons that reside in An. gambiae (Grossman et
al., 1999). The term ‘element’ is used interchangeably with ‘transposon’. After an
element invades a genome, it multiplies, resulting in multiple copies. Amplification of
DNA TEs is tied to DNA replication and host repair. Copies are referred to as full-length
or truncated. Full-length copies contain all features of the transposon, including TIRs and
an intact open reading frame (ORF). It is assumed that full-length copies are potentially
autonomous, that is, retain the ability to move and be moved. Truncated copies are those
missing portions of the transposon, terminally, internally, or both. Those that retain intact
TIRs retain the ability to be moved by their cognate transposase in trans. Truncated
copies are also referred to as non-autonomous.
1.1.3 IS630-Tc1-mariner Superfamily of Class II Transposable Elements
The focus of this dissertation is the DD34E TEs from the IS630-Tc1-mariner
superfamily. Representatives from this superfamily are found in a wide range of
organisms including bacteria, plants, fungi and animals, and are probably the most
widespread DNA TEs in nature (Plasterk & van Luenen, 2002) and references therein).
There is substantial evidence for horizontal transfer for some members of this
superfamily, particularly for the mariner element (summarized in Robertson et al., 2002),
and this process has probably contributed to their widespread distribution and
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evolutionary success (Robertson & Lampe, 1995b). These elements typically range from
1.3 to 2.4 kilobase pairs (kb) in length, are flanked by TIRs typically 20-500bp in length,
and contain a single gene encoding a transposase (Plasterk et al., 1999). They target ‘TA’
sequences for integration into the host genome, resulting in ‘TA’ target-site duplications
flanking the integrated transposon (van Luenen et al., 1994).
The catalytic domain of this superfamily contains a conserved motif of
D(Asp)DE(Glu) or DDD triad, which has been shown to be necessary for transposition
activity through in vivo experimentation (Lohe et al., 1997). The triad coordinates
divalent metal ions which are thought to assist nucleophilic attacking groups during the
cleavage and strand transfer reactions during transposition, and is found in the integrase
enzyme of some RNA elements (Class I elements) and retroviruses, thus suggesting a
possible common origin between these elements and members of the IS630-Tc1-mariner
superfamily (Capy et al., 1996).
The members of the IS630-Tc1-mariner superfamily can be further classified
based on the number of amino acid residues between the second D and the third D/E
residue of the catalytic triad (Shao & Tu, 2001). For example, all Tc1-like elements
contain a DD34E motif in which 34 amino acids separate the second D from the third E
residue. To date, the “triads” that have been identified in An. gambiae are DD34D,
DD34E, DD37D and DD37E (Robertson, 1993; Grossman et al., 1999; Shao & Tu, 2001;
Shao and Tu, unpublished). IS630-Tc1-mariner members containing variable numbers of
amino acid residues between the second and third amino acids (DDxD) have also been
identified, namely those belonging to the pogo family of TEs (Shao & Tu, 2001).
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The N-terminal domain of Tc1-mariner transposases is involved in the
recognition and binding of its cognate TIRs (reviewed in Plasterk et al., 1999). Binding is
mediated by two helix-turn-helix (HTH) motifs, designated the PAI and RED domains (N
and C-terminus within the N-terminus, respectively). They, in turn, are connected by a
short stretch of conserved amino acids, the GRPR sequence, which is thought to stabilize
DNA binding (Izsvak et al., 2002; Yant et al., 2004). These two HTH motifs occupy
approximately the first 120 amino acid residues in the transposase. Through binding
assays (Yant et al., 2004) and crystallographic studies (Watkins et al., 2004), it has been
shown that the recognition and binding are primarily mediated by the PAI domain in the
Tc1 transposons. The RED domain may play a role in stabilizing the DNA/transposase
complex through non-specific DNA binding (Vos et al., 1993; Colloms et al., 1994;
Pietrokovski & Henikoff, 1997; Watkins et al., 2004).
1.1.4 Evolutionary Impact of Transposable Elements on Genome Function and
Evolution
The ways in which TEs are believed to have influenced genome function and
evolution can be broken down into three main categories: 1) Molecular domestication,
whereby the genome has co-opted the activity of TEs to perform functions within the
genome; 2) Accelerated evolution, whereby TEs can bring about gross changes within the
genome; 3) Evolutionary drive, whereby the host and TE engage in an evolutionary
“arms race”.
Throughout evolutionary history, the activity of TEs has been harnessed by and
for the benefit of the genome. A familiar case is telomere maintenance in Drosophila in
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which the non-LTR retrotransposons, TART and HeT-A, maintain telomeres rather than
telomerase as in other eukaryotes (Biessmann et al., 1992; Levis et al., 1993). Strikingly,
it appears that these two TEs, despite their separate evolutionary origins, work
cooperatively, rather than competitively, to maintain telomeres (Pardue & DeBaryshe,
1999). The classic example of molecular domestication, however, is the RAG1/RAG2
enzyme system responsible for the recombination of the V(D)J locus in the vertebrate
adaptive immune system. These enzymes are believed to have evolved from an ancient
DNA TE, and show remarkable functional and structural similarities to the Tc1 family
(reviewed in Miller & Capy, 2004).
Some researchers argue that transposable elements provide a means for the
rapid evolution of proteins by virtue of their ability to produce gross changes within the
genome unattainable via simple random drift (reviewed in Kidwell & Lisch, 2001). It has
been suggested that the genome can take advantage of the gross genomic changes
generated and facilitated by TEs to better adapt the host to stressful environmental
conditions (Wessler, 1996). It is argued by some researchers that “TE activity can be seen
as an essential component of the hosts’ response to stress, facilitating the adaptation of
populations and species facing changing environments” (Ashburner et al., 1998). This
notion is supported by the increase of TE activity in response to abiotic and biotic
stressors (reviewed in Wessler, 1996). Unlike molecular domestication, unambiguous
examples of accelerated evolution are few; however chromosomal inversions constitute
an excellent example of how TEs can facilitate rapid change. Transposable elements have
been shown to be associated with the break-points of chromosomal inversions in fly
species, including hobo in Drosophila (Miller & Capy, 2004) and Odysseus in
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(Mathiopoulos et al., 1998; Mathiopoulos et al., 1999). Chromosomal inversions in
Anopheles species have been associated with better adaptation of species to their
particular environments through the new gene linkages created by the inversions (Powell
et al., 1999; Coluzzi et al., 2002; Ayala & Coluzzi, 2005). It appears that TEs may have
played an important, albeit inadvertent, role in better adapting these mosquitoes to their
environment.
Perhaps the most controversial theory concerning TE influence in genome
evolution is the hypothesis that mechanisms which evolved within the host genome to
control TE activity have subsequently lead to the evolution of more complex organisms,
much like technology that comes out of an arms race (McDonald, 1998). Specifically, it
is proposed that TEs were instrumental in the prokaryotic/eukaryotic and the
invertebrate/vertebrate macro-evolutionary transitions (McDonald, 1998). Chromatin
formation and methylation, mechanisms believed to be prerequisites for the
prokaryotic/eukaryotic and invertebrate/vertebrate transitions, respectively (Bird, 1995),
are hypothesized to have originally evolved as mechanisms to squelch TE activity by the
genome (McDonald, 1998).
With each newly sequenced genome, becomes increasingly apparent that TEs
have played a larger role in genome evolution than previously thought, and as a
consequence, the paradigm for TEs is changing. A deeper look at TEs and their
relationships with their hosts reveals a subtle interplay that defies a single definition for
these genetic elements. It appears that each TE-host case is dependent upon the individual
relationship between the two and will have to be evaluated independently to determine
where the TE falls in the continuum of parasite to benefactor (Kidwell & Lisch, 2001).
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1.1.5 Horizontal Transfer of Transposable Elements
Horizontal transfer refers to the processes by which genetic material crosses species
boundaries to invade a naïve genome. The mechanisms by which transposable elements
undergo horizontal transfer remain largely unknown. However, there is evidence that it
may be mediated by the movement of viruses, as in the case of the Lepidopteran DNA
TE piggyBac (Fraser et al., 1983; Cary et al., 1989; Zimowska & Handler, 2006);
introgressive hybridization as suggested for the distribution of P elements among some
Drosophila species (reviewed in Silva & Kidwell, 2000); or through the intimate
relationship between host and parasite, as in the case of the mariner element shared
between the parasitoid wasp Ascogaster reticulates and its host moth Adoxophyes honmai
(Yoshiyama et al., 2001).
Past horizontal transfer events are typically inferred from three types of
circumstantial evidence (reviewed in Silva et al., 2004). The most persuasive cases of
horizontal transfer demonstrate all three. The first indicator is high sequence identity of
the TE between divergent species, in which case the identity of the TE exceeds that of
most host genes. The second is incongruence between the TE phylogeny and that of the
host species in which the TE is found. Finally, patchy distribution of the TE among
closely related species can also suggest horizontal transfer, although it remains a
possibility that the TE was lost from the species in which the element is absent.
Several cases of horizontal transfer of elements in the IS630-Tc1-mariner
superfamily are known, with the strongest cases for mariner elements (Robertson et al.,
2002). All three pieces of evidence have been shown for mariner, and horizontal transfer
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of this element has been well demonstrated (reviewed in Robertson & Lampe, 1995a). It
is hypothesized that horizontal transfer is an essential step in the lifecycle of Tc1-mariner
elements. Existing evidence suggests that horizontal transfer is the only step wherein
selection occurs during their lifecycle (Lampe et al., 2003). These elements do not
require host factors for transposition, and therefore are not restricted with regard to the
genomes which they can invade. This characteristic has probably contributed to their
success in invading a wide diversity of organisms (Robertson & Lampe, 1995b). Other
examples of horizontal transfer within this superfamily include Minos (Arca & Savakis,
2000; de Almeida & Carareto, 2005) and TCp3.2 (Jehle et al., 1998), both of which are
Tc1 elements.
1.1.6 Utility of Transposable Elements as Biological Tools
In addition to being important components of genomes, transposable elements have
proven to be valuable biological tools. They are used as genetic markers in population
studies (reviewed in Tu & Li, 2005), in the exploration of genomes by promoter and gene
trapping (e.g., Springer, 2000), and to introduce transgenes into genomes (reviewed by
Handler & O’Brochta, 2005). Because they can rapidly spread through a population, they
have been proposed as drive mechanisms to drive transgenes (Carareto et al., 1997). The
utility of a given transposon for transgenesis depends on the endogenous TEs residing in
the genome to be manipulated, and what host factors, if any, are required for its mobility.
Cross-reactivity can occur between endogenous and exogenous TEs (Sundararajan et al.,
1999; Jasinskiene et al., 2000), potentially leading to uncontrolled, unwanted
transposition events. Problems can also arise if the host genome cannot provide necessary
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factors for transposition. This can result in either no transposition activity or
uncontrolled, indiscriminate activity, both of which are usually undesired (O'Brochta &
Atkinson, 1996). Identification of the TEs resident within a genome will enable better
decision-making in selecting TEs for transgenesis.
1.1.7 Identification of Active Elements
Aside from their use as genetic markers, the utility of transposable elements as tools
requires them to be active or functional. Active TEs can be identified with varying levels
of confidence by a number of means. Most active TEs have been identified from the
spontaneous mutations they cause in the host organism, such as Ac from Zea mays
(McClintock, 1948) and Tc1 from Caenorhabditis elegans (Emmons et al., 1983).
However, this approach depends on the serendipity of finding such an element in the
organism of interest. Therefore circumstantial evidence is often used when an active TE
is being sought. Examples of such evidence include high degree of polymorphism of a
TE between individuals of the same species. Because such an observation can also result
from ancestral polymorphism, corroborating evidence is necessary before any final
interpretation can be made. This might include the TE being in one species or in high
copy number, but not in another, closely related species, or the identification of footprints
left behind after the excision of the TE within the host genome (evidence of past
mobility). Stronger evidence of activity includes the identification of transcripts from the
TE, or detection of extrachromosomal copies of the TE in the process of transposition by
Southern blot or nested, inverse PCR. Finally, TEs can be tested for their ability to carry
out transposition in a number of in vitro and in vivo transposition assays. It is important
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to note that although a TE may be able to carry out transposition; this does not
necessarily mean that it is active, just functionally competent. It is also important to bear
in mind that differences have been observed in TE activity depending on the nature of the
system in which the element is being tested (e.g. Pledger et al., 2004), suggesting that TE
behaviour is species-dependent, and activity in one system is not a guarantee of activity
in another.
1.2 Mosquitoes 1.2.1 Anopheles gambiae
The principal organism targeted by this project is the African malaria mosquito,
Anopheles gambiae. An. gambiae, which is highly anthropophilic in nature, is the primary
vector of the malaria parasite, Plasmodium falciparum. P. falciparum is the most lethal of
four Plasmodium species that causes malaria in humans. An. gambiae is also a vector of
other important diseases including filariasis, caused by the nematode Wuchercheria
bancrofti, and O’nyong-nyong Fever, caused by an arbovirus. The An. gambiae genome
sequence, which was released in 2002 (Holt et al., 2002), is approximately 278 million
base pairs (Mbp) in size. It is estimated that 16% of the euchromatic and 60% of the
heterochromatic region is comprised of transposable elements. A number of Tc1
transposable elements from An. gambiae have been described (Grossman et al., 1999;
Hill et al., 2001; Warren, et al., unpublished data), but with the availability of the An.
gambiae genome it became possible to conduct a systematic, genome-wide search and
analysis of these elements. While An. gambiae was the focus of this project, other
mosquito species were included. Their relationships to An. gambiae are described below.
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1.2.2 Mosquito Taxonomy and Taxonomic Relationships
Mosquitoes are dipteran insects, also known as “true flies”, belonging to the family
Culicidae. They can be found all over the world, with exception to those places that are
permanently frozen (Clements, 1992). Three subfamilies of mosquitoes exist, the
Culicinae, Anophelinae, and Toxorhynchitinae, the first two of which contain important
mosquito species involved in disease transmission.
An. gambiae is a member of the Anopheles gambiae species complex, which is
comprised of at least seven morphologically indistinguishable species (Coetzee et al.,
2000 and references therein). The complex also includes An. arabiensis, An.
quadriannulatus A and B, An. bwambae, An. merus and An. melas, all of which belong to
the subfamily Anophelinae. Hybrids have been observed in nature between An.
arabiensis and An. gambiae, An. arabiensis and An. quadriannulatus, and An. gambiae
and An. melas (Powell et al., 1999 and references therein). However hybrids are rare,
accounting for less than 0.1% of populations sampled (Coluzzi et al., 1979; Petrarca et
al., 1991; Toure et al., 1998). Most experimental crosses between these species produce
fertile F1 females but sterile F1 males with the exceptions that crosses between An.
quadriannulatus females against An. gambiae males and An. quadriannulatus females
against An. bwambae males yield some fertile F1 males as well as females (Powell et al.,
1999 and references therein). In addition, there is growing evidence that suggests
introgression occurs between An. gambiae and An. arabiensis (Besansky et al., 1994;
Powell et al., 1999; Besansky et al., 2003; Donnelly et al., 2004). These results suggest
that these species retain enough similarity between their genomes for gene flow to occur,
thereby providing an avenue for horizontal transfer, an important consideration when
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contemplating the introduction of transgenes. The species An. gambiae itself can be
divided into two genetically distinct variants, the molecular forms M and S (della Torre et
al., 2001). These two variants can interbreed to produce viable offspring, however,
hybrids are rarely found in nature. Therefore, it is hypothesized that these two forms are
undergoing insipient speciation (della Torre et al., 2001).
In addition to members of the An. gambiae species complex, other Anopheline
and non-Anopheline mosquitoes were used in this study in order to compare the different
magnitudes of evolution within various mosquito lineages. The Anophelines included
were, in order of increasing divergence from An. gambiae, An. stephensi, An. dirus and
An. faraurti, and An. albimanus. Mosquitoes outside of the Anophelines included
members of the Culicinae subfamily, Aedes aegypti, Ae. albopictus, Ochlerotatus
atropalpus, and Culex pipiens quiquefasciatus. The divergence time estimated between
An. gambiae and An. stephensi is 5-15 million years ago (MYA) (Gaunt & Miles, 2002)
as compared to the estimated 145-200 MYA between An. gambiae and Ae. aegypti
(Krzywinski et al., 2006). In addition to An. gambiae genome, there were two other
mosquito genomes available for comparative analysis, Aedes aegypti (Subfamily
Culicinae, approximate genome size 1300Mbp), and Culex pipiens quinquefasciatus
(Culicinae, 540Mbp). Aedes aegypti is an important disease vector involved in the spread
of yellow fever. A second draft assembly of this genome has been released (Nene, et al.,
2007). Cx. pipens is a widespread mosquito species involved in the transmission of West
Nile virus. This genome project is in its early stages, and during the course of this work,
was comprised of trace reads (ftp://ftp.ncbi.nih.gov/pub/TraceDB/).
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1.2.3 Controlling Disease through Genetic Manipulation Insecticides have played a major role in the control of mosquitoes and, by extension,
malaria. However, because of growing insecticide resistance among mosquitoes,
alternative methods of pest control are being sought, such as the development genetically
modified mosquitoes that are refractory to Plasmodium or have reduced capabilities to
transmit the parasites to human hosts. One of the proposed mechanisms for transforming
and driving transgenes into mosquito populations involves the use of TEs.
The concept of the TE-mediated genetic transformation of mosquitoes is
straightforward. Once a gene and appropriate control regions are identified, they are
incorporated into a plasmid construct, along with the coding region of the transposase of
the TE being used, flanked by the cognate TIRs recognized by the TE. This construct is
microinjected into embryos, where the transposase is expressed, and transposes the entire
construct inserted into the genome. Transformants are chosen using a selection marker
that is also included in the construct. The TE chosen for transformation will both
introduce transgenes into the genome and subsequently drive the transgene into the
mosquito population. In theory this approach is simple, and great strides have been made
towards this end. However, there are a number of technical and ethical issues remain to
be resolved.
Low embryonic survival from the microinjections has hindered transformation
in some mosquito species (reviewed in Atkinson et al., 2001). The ratio of transgenic
mosquitoes to the number of injected eggs is often low, and performing injections is
laborious and tedious. In addition, a reduction in fitness is often observed in transgenic
organisms, rendering them less competitive than their wild-type counterparts (reviewed
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in Marrelli et al., 2006). Transgene products can be toxic, especially at high levels, and
disruption of host genes or regulatory regions can occur upon insertion of the TE
(Marrelli et al., 2006). However, the use of tissue and temporal specific promoters (e.g.
Moreira et al., 2000) and the development of chimeric proteins designed to target specific
DNA sequences show promise in overcoming these obstacles (Maragathavally et al.,
2006).
Transgene expression can be influenced by the regulatory sequences upon
insertion surrounding the insertion, sometimes resulting expression levels can be
undesirable or insufficient. To circumvent this problem, insulators are being designed to
buffer the internal gene (Sarkar et al., 2006). However, implementation of this approach
is somewhat constrained by the limited carrying capacity of some TEs (e.g. Lampe et al.,
1998; Fischer et al., 1999; Karsi et al., 2001). Problems involving the integration of TEs
have also observed, such as with Hermes, which often includes flanking DNA from the
plasmid construct (reviewed in O'Brochta et al., 2003), and piggyBac, which sometimes
remobilizes by mechanisms other than cut-and-paste, resulting in multi-copy insertions in
the form of tandem arrays of the element (Adelman et al., 2004). However, the most
problematic obstacle yet to be overcome is poor post-integration mobility of the TE-
transgene in the germ line of transformed mosquitoes (reviewed in O'Brochta et al.,
2003). While a handful of DNA TEs are successfully utilized in primary transformation
of mosquitoes, including Minos, Mos1, Hermes, and piggyBac, all exhibit little or no
post-integration mobility in the germ line (reviewed in O'Brochta et al., 2003).
The intrinsic attributes of TEs make them extremely attractive as biological tools
– their invasiveness, the structural and functional consequences of their genomic
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insertions, and their potential to cross species boundaries (Miller & Capy, 2004).
However, these features also make TEs potentially dangerous for transgenic applications.
A better understanding of natural TE behaviour in mosquitoes will facilitate overcoming
the aforementioned obstacles and help eliminate unwanted consequences of their use in
transgenesis. This dissertation project will directly impact that effort by examining the
diversity, behaviour, and evolution of DD34E TEs in the An. gambiae genome, as well
their impact upon genome evolution.
1.3 Dissertation Scheme 1.3.1 Research Aims, Scheme, and Significance
This dissertation project was comprised of three distinct, but interrelated aims, designed
to maximize the chances of identifying a functional transposase that could then be studied
further to better understand TE behaviour in natural mosquito populations. The aims were
as follows:
• Aim 1: Complete a genome-wide, systematic survey of the DD34E
transposable elements in the published genome of Anopheles gambiae. Select
one or a few DD34E transposable elements with characteristics of recent
activity for further study.
• Aim 2: Determine the distribution of selected DD34E TE(s) in natural
populations of the An. gambiae species complex and other mosquito species.
• Aim 3: Investigate the potential transposition activity of selected DD34E TEs
in an in vitro inter-plasmid assay system.
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CHAPTER 2
Gambol and Tc1 are two distinct families of DD34E transposons: Analysis of the
Anopheles gambiae genome expands the diversity of the IS630-Tc1-mariner
superfamily
Coy and Tu, 2005
Permission to reprint granted by Blackwell Publishing Company
Insect Molecular Biology
Volume 14 Issue 5 Page 537 - Oct 2005
2.1 Abstract
Tc1 is a family of DNA transposons found in diverse organisms including
vertebrates, invertebrates and fungi. Tc1 belongs to the IS630-Tc1-mariner superfamily,
which are characterized by common ‘TA’ target site and conserved D(Asp)DE(Glu) or
DDD catalytic triad. All functional Tc1-like transposons contain a transposase with a
DD34E catalytic triad. We conducted a systematic analysis of DD34E transposons in the
African malaria mosquito, Anopheles gambiae, using a reiterative and exhaustive search
program. In addition to previously described Tc1-like elements, we uncovered 26 new
DD34E transposons including a novel family that we named gambol. Designation of
family status to gambol is based on phylogenetic analyses of transposase sequences that
showed gambol and Tc1 transposons as distinct clades that were separated by mariner
and other families of the IS630-Tc1-mariner superfamily. The distinction between Tc1
and gambol is also consistent with the unique TIRs in gambol elements and the presence
of a ‘W[I/L/V]DEDC’ signature near their N-termini. This signature is predicted as part
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of the ‘RED’ domain, a component of the ‘PAI’ and ‘RED’ DNA binding domains in Tc1
and possibly mariner. Although gambol appears to be related to a few DD34E
transposons from cyanobacteria and fungi, no gambol has been reported in any other
insects or animals thus far. Several gambol and Tc1 elements have intact ORFs and
different genomic copies with high sequence identity, which suggests that they may have
been recently active.
2.2 Introduction
IS630 from bacteria, Tc1 from worm, and mariner from fly are the founding
members of the IS630-Tc1-mariner (ITm) superfamily, a group of DNA transposons
found in a wide diversity of organisms from bacteria to vertebrates (Doak et al., 1994;
Shao & Tu, 2001). These transposons contain a single gene encoding a transposase,
which is flanked by terminal inverted repeats (TIRs) that specify their 5’ and 3’
boundaries (Plasterk et al., 1999). They target ‘TA’ sequences for integration into the
host genome, resulting in ‘TA’ target-site duplications (TSDs) flanking the integrated
transposon (van Luenen et al., 1994). Many elements in this superfamily range between
1.3 – 2.4 kb in length although there are examples of longer elements. The catalytic
domain of the transposase is located in the C-terminus, and contains a conserved
D(Asp)DE(Glu) or DDD triad, which has been shown to be necessary for transposition
(Lohe et al., 1997). The distances between the first two D’s are variable while the
distances between the last two residues in the triad are conserved within Tc1 and mariner
families. Not counting the degenerate copies, all Tc1-like transposons are characterized
by a DD34E catalytic triad and all mariner-like transposons are characterized by a
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DD34D catalytic triad. The Arabic numeral here indicates the number of amino acids
between the second and third residues of the triad.
Phylogenetic analyses based on transposase sequences from diverse organisms
suggest that IS630-Tc1-mariner transposons comprise at least six families in eukaryotes:
Tc1, mariner, ITmD37D, ITmD39D, ITmD41D, and ITmD37E (Doak et al., 1994;
Gomulski et al., 2001; Shao & Tu, 2001, Robertson, 2002). As shown for Tc1 and
mariner, the above phylogenetic classification is consistent with the profile of the
transposase catalytic triad in all six families. Therefore ITmD37D, ITmD39D, ITmD41D
and ITmD37E represent distinct families of transposons that contain DD37D, DD39D,
DD41D, and DD37E catalytic triads respectively. ITmD37D and ITmD39D were
previously considered to be basal subfamilies of mariner elements, namely the Bmmar1
and Soymar1 subfamiles (Robertson & Asplund, 1996; Jarvik & Lark, 1998). The
congruence between phylogenetic classification based on transposase sequences and the
classification according to the catalytic triad profile reflects the functional importance of
the triad in IS630-Tc1-mariner transposons. However, there are two noted exceptions
(Shao & Tu, 2001). First, pogo-like elements are characterized by the DDxD triad where
“x” indicates variable number of residues. Pogo has a unique N-terminal DNA-binding
domain and a long C-terminal domain rich in acidic residues. Pogo may either be a
highly divergent group within the IS630-Tc1-mariner superfamily, or a related
superfamily. The second exception refers to the phylogenetically unresolved elements
that comprise the DDxE transposable elements (TEs). They include DDxE transposons in
bacteria and archea and a few DD34E transposons from ciliates and fungi (Shao & Tu,
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2001). These DDE transposons are phylogenetically distinct from the Tc1 family that also
contains a DD34E catalytic triad.
Transposons in the Tc1 family are widely distributed in vertebrates, invertebrates,
and fungi. Examples include SleepingBeauty, an active element reconstructed from
several inactive copies from fish genomes (Ivics et al., 1997); Tc3, a large group found in
nematodes and insects (Collins et al., 1989; Tu & Shao, 2002); and Impala, a divergent
member in a soil-borne fungus (Langin et al., 1995). In addition to the catalytic domain
that is characterized by the DD34E triad, Tc1 transposases contain a N-terminal region
that is involved in the recognition and binding of the transposase to their TIRs (reviewed
in Plasterk et al., 1999). These binding activities are mediated by two helix-turn-helix
(HTH) motifs, namely the PAI and RED domains (N and C-terminus, respectively) that
are connected by a short stretch of amino acids (Izsvak et al., 2002; Yant et al., 2004).
These two HTH motifs occupy approximately the first 120 amino acid residues in the
transposase. Through binding assays (Yant et al., 2004) and crystallographic studies
(Watkins et al., 2004), it has been shown that the recognition and binding is primarily
mediated by the PAI domain in the Tc1 transposons. The RED domain may play a role in
stabilizing the DNA/transposase complex through non-specific DNA binding (Vos et al.,
1993; Colloms et al., 1994; Pietrokovski & Henikoff, 1997; Watkins et al., 2004). In
contrast, the entire region of the N-terminal 140 residues of a mariner transposon Mos1 is
required for efficient and specific binding to its TIRs by the transposase, which led to the
suggestion that the N-terminal region of mariner-like elements is of different origin than
that of the Tc1-like elements (Auge-Gouillou et al., 2001).
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Here we report a systematic analysis of all DD34E transposons in the genome of
the African malaria mosquito, Anopheles gambiae. In addition to the previously
described Tc1-like elements (Grossman et al., 1999; Hill et al., 2001), we uncovered 26
new DD34E transposons including a novel group that we named gambol. The name is
derived from gambiae and it also means to “leap about playfully”. Although gambol
elements contain a DD34E catalytic triad, they are distinct from Tc1 transposons
according to phylogenetic analysis of their transposase sequences and additional
supporting evidence. Gambol appears to be more closely related to a few DD34E
transposons from cyanobacteria and fungi, and it represents a new family in the IS630-
Tc1-mariner superfamily.
2.3 Materials and Methods
2.3.1 Identification of DD34E TEs in An. gambiae
The An. gambiae genome assembly (Holt et al., 2002) consisting of 8987
scaffolds was downloaded from NCBI on July 30, 2003. Forty-nine full length amino
acid sequences representing major clades within the IS630-Tc1-mariner superfamily were
used as queries to identify DD34E transposons in An. gambiae. Sequences from groups
other than DD34E were included to insure comprehensive searching of the database, and
to identify potentially distant DD34E members.
The search strategy followed that described by Biedler & Tu (2003), using a
multi-query Blast search (Altschul et al., 1997) and the computer programs described
therein (TEpost, TEcombine, FromTEpost, and TEmask). These programs are serially
linked together so that the output of one is used as input for the next. The series of
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programs is run as one automated program, called TEpipe. The strategy is briefly as
follows: The above query sequences were blasted against the An. gambiae database
(chopped up into 100 kb fragments to speed up the search) using a tBlastN search with an
E-value cutoff 1e-5. TEpost organized the results from this search and TEcombine
removed redundant hits. The output from TEcombine was used by FromTEpost to
generate a non-redundant list of putative IS630-Tc1-mariner TEs in fasta format with
scaffold positions. The longest sequence from this list was then used as a query in a
BlastN search (E-value: 1e-10) against the An. gambiae database. The results of the
BlastN search were then used as input for TEpost, TEcombine, and FromTEpost as
described above, resulting in a list of sequences in fasta format that represented putative
copies that belong to the same transposon as the query copy. TEmask masked all copies
of a TE from the database, and the search process was repeated until no more potential
elements were identified. TEpipe provided an alignment of the transposon members
using ClustalW (v. 1.81 for Linux, default settings) (Thompson et al., 1994). In essence,
TEpipe rapidly produced a list of candidate TEs of interest that are subject to further
phylogenetic analysis and manual inspection to verify/clarify the classification and
boundaries of each TE. Under the settings described above, each transposon is
comprised of copies that are less than 20% divergent from each other. Chopping up the
An. gambiae database did not affect the overall coverage of the survey as no match was
found at the boundaries of the fragments. TEpipe and individual programs can be
downloaded from http://jaketu.biochem.vt.edu/dl_software.htm. TEpipe was run on a
Dell 530 Linux workstation with twin 2.0 GHz processors, 1.5 Gb RAM, and 80 Gb hard
drive.
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2.3.2 Characterization of DD34E TEs in An. gambiae
Alignments generated with ClustalW for Linux v. 1.81 (Thompson et al., 1994)
and/or ClustalX for Windows v. 1.83 (Thompson et al., 1997) were used to identify TE
boundaries and TSDs for each transposon. For some TEs, there was only one potentially
full-length member, in which case alignments could not be used to determine TE
boundaries. In these cases the sequences were run through EINVERTED (EMBOSS:
http://ngfnblast.gbf.de/cgi-bin/emboss.pl?_action=input&_app=einverted) in order to
locate putative TIRs, which for several transposons, led to the identification of their
boundaries and TSDs. Translations were predicted using the Translation Machine at
EBI: http://www2.ebi.ac.uk/translate/. TIR consensus sequences were generated using
WebLogo (http://weblogo.berkeley.edu/logo.cgi) (Schneider & Stephens, 1990; Crooks
et al., 2004). Helix-turn-helix motifs were predicted with PROF predictions (Rost &
Sander, 1993; Rost et al., 1996) at http://www.predictprotein.org.
2.3.3 Copy number, genomic distribution, and functional classification of DD34E
transposons
To determine copy number, a full-length member (intact ORF and TIRs) was
chosen as a representative and used as a BLASTn query (E-value: 1e-10) against the An.
gambiae genome. For TEs that did not have a full-length representative, the longest
member was used as the query sequence. The BLAST results were run through TEpost,
from which the copy number was determined. All copies of a TE have sequence
identities above 80%, and most are above 90%, to the query sequence. TEs were then
classified according to the following characteristics: 1) TEs that had TIRs and intact
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ORFs coding for a transposase were labeled as potentially ‘autonomous’. These are
elements that hypothetically could either mobilize themselves or other members of the
family with intact TIRs. 2) Those with intact TIRs but not ORFs were categorized as
‘non-autonomous with TIRs’. These hypothetically retain the ability to be recognized by
the appropriate transposase, and therefore could be mobilized. 3) TEs that no longer have
intact TIRs and are therefore incapable of moving themselves or being moved by
autonomous elements were classified as ‘non-autonomous without TIRs’. If a TE had no
TIRs, but did have an ORF, they were placed in this category. A number of hits were
rejected and not counted towards copy number, most of which were short matches (on
average less than 100bp) at the extreme 5’ or 3’ end of the query sequence. Hits that
corresponded to matches against an inserted element within the query sequence (i.e., a TE
within the TE of interest) were rejected as well. Chromosomal locations of TEs were
determined through the use of TEMap (Mao & Tu, unpublished), which converts the TE
positions from the TEpost output to chromosomal locations and allows for determination
of TE density and their correlations with gene density. The An. gambiae genome
assembly maintained and updated by the ENSEMBL (EBI) site was downloaded and
used for this analysis.
2.3.4 Phylogenetic Analysis
Neighbor-joining (NJ) and maximum-parsimony (MP) analyses, as implemented
by PAUP* v. 4.0 b10 for Windows (Swofford, 2003), was used to infer phylogenetic
relationships between the An. gambiae DD34E and representative IS630-Tc1-mariner
transposase sequences. MP analysis was conducted using the heuristic search algorithm
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with 100 random sequence additions and tree-bisection-and-reconnection branch
swapping. Bootstrapping was used to determine confidence of groupings with 2000
replicates for both NJ and MP methods. Whole transposase sequences were used for
phylogenetic analyses unless otherwise specified. Alignments of the transposase
sequences were generated with ClustalX v. 1.83 (Thompson et al., 1997). Alignment
parameters used were a gap opening penalty of 5.0 and a gap extension penalty of 0.5.
Minor adjustments to alignments, when necessary, were done with SeaView (Galtier et
al., 1996). TreeView was used to visualize the resulting trees (Page, 1996).
2.4 Results
2.4.1 Identification of twenty-six new DD34E transposons in An. gambiae
Our systematic survey, using a computer program called TEpipe, identified 26
new DD34E transposons in An. gambiae, in addition to all previously reported DD34E
elements. Among these, AgH1 was found prior to this analysis during a search for the
DD37D and DD37E transposons in An. gambiae (Table 2.1; Seok, Shao & Tu,
unpublished). Fourteen of the 26 new elements have members with TIRs and/or intact
ORFs, nine of which have both. Table 2.1 lists the names, classification, characteristics,
and copy number of these 14 new TEs, along with the previously identified An. gambiae
DD34E elements. The remaining 12 elements are highly degenerate with no TIRs, and
were not characterized further. All identified TEs have been reported to ENSEMBL
Mosquito gene and TE pages: http://www.ensembl.org/Anopheles_gambiae/submission.
Conceptual translations of the DD34E TEs produce proteins ranging in size
between 330-374 amino acids. There is one exception, Lovejoy (Table 2.1), which
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encodes for 236 amino acids. While this element has an intact ORF, it is unknown
whether the protein it codes for would be capable of carrying out transposition. A
transposase of 236 amino acids would be the shortest DD34E transposase reported to
date, although it is possible that it could result from truncation (see discussion below).
Two of the 14 new elements that are characterized are Tc1-like, one of which is a
degenerate homologue of Tc3, while the remaining 12 TEs form a novel group of DD34E
transposons that we named gambol. As described later, gambol is a novel transposon
family in the IS630-Tc1-mariner superfamily.
2.4.2 Topi, a previously identified Tc1 element, is comprised of three lineages
Topi is a member of the Tc1 family, and was identified by Grossman et al. (1999)
through the use of degenerate primers and genomic library screening. They reported two
copies of Topi, one full length and one truncated copy, which they named Topi I and Topi
II, respectively. During our analysis of the multiple sequence alignment of Topi elements,
it became apparent that Topi comprises three lineages which we named Topi.1, 2 and 3.
The division of Topi elements is supported by distance data, where the average identity of
copies within lineages (98%) is greater than between lineages (91%). The TIRs of the
lineages also differ slightly, particularly at the 5’ end (Table 2.1). According to our
analysis, the two copies identified by Grossman et al. are both members of Topi.1. Topi is
among the most abundant DD34E transposons in An. gambiae, second only to Tsessebe,
another Tc1-like transposon (Grossman et al., 1999). High sequence identity between
members of Topi.1, up to 100%, suggests that these elements may have been recently
active.
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2.4.3 The gambol elements
According to the alignment of the transposase sequences, twelve of the new
DD34E transposons form the distinct gambol group (Fig. 2.1A). The amino acid
sequence similarity between members of this group ranges from 38 to 68%. Most of
these elements share a conserved ‘W[I/L/V]DEDC’ (Trp-[Ile/Leu/Val]-Asp-Glu-Asp-
Cys) amino acid signature, approximately 80 amino acids upstream from the catalytic
domain (Fig 2.1A and B). A similar signature is found in similar positions within the
ORFs of two mariner (DD34D) TEs, M.destructor.mar1 and D.mauritiana.mar1, with
signatures LLDEDC and LLDEDD, respectively (Fig 2.1A). A similar signature was also
found in two previously unidentified mariner elements in An. gambiae (LLDEDD and
LLDEDS, data not shown). Although BLAST searches using the gambol sequences
produced hits against known or putative TEs, no TEs with this signature were identified
in any other organism. Figure 2.2 shows that the W[I/L/V]DEDC signature is located in
the predicted RED domain of the gambol elements, making up the C-terminal end of first
helix and the remainder residing between the first and second helix of that domain. While
the predicted secondary structures are highly conserved between different gambol
elements, their overall primary sequences are not except for the W[I/L/V]DEDC
signature, suggesting that this primary sequence may be important in the function of the
transposase. A few of the gambol elements are not represented by potentially autonomous
members (Table 2.1). Therefore these elements are probably no longer active, which is
why they either lack the conserved W[I/L/V]DEDC signature or the DD34E triad.
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2.4.4. Phylogenetic analyses place gambol elements in a distinct group separate from
Tc1 transposons
The inferred phylogenetic relationships between the An. gambiae DD34E
elements and representative transposases from the IS630-Tc1-mariner superfamily are
depicted in Figure 2.3 as an unrooted NJ phylogram. The major families of the
superfamily described in Shao and Tu (2001) form their respective clades. Although
gambol and Tc1 are all DD34E transposons, our analysis places gambol and Tc1
transposases in distinct groups that are supported by high bootstrap values (Fig 2.3). We
tested the robustness of the relationships shown in Figure 2.3 using different phylogenetic
treatments. MP analysis with the same alignment produced a single tree with a similar
topology, differing only in the placement of the DD39D TEs (ITmD39D). MP places
them as a sister group to the DD34D TEs (mariner), although this placement was not
supported by bootstrap analysis. The other differences were minor changes in the
placement of transposons within the Tc1 and gambol groupings. Removal of apparently
non-functional gambol elements (Table 2.1) did not change the overall tree topology in
NJ and MP analyses, neither did the addition of diverse IS630-Tc1-mariner elements
used in the analysis by Shao and Tu (2001) (data not shown). All above phylogenetic
analyses indicate that there are several levels of groupings separating the gambol and Tc1
clades. In other words, gambol and Tc1 are separated by mariner (DD34D) and other
transposon families in the IS630-Tc1-mariner superfamily. For example, Tc1 elements
are more closely related to the DD37D clade than any other clades (Shao and Tu, 2001)
including gambol. No matter where one roots the tree, Tc1 and gambol are two distinct
clades. Therefore we felt justified to designate gambol as a transposon family
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independent of Tc1, mariner and other families (e.g., DD37D, DD37E) in the IS630-Tc1-
mariner superfamily.
Both NJ and MP methods suggest that gambol elements may be related to some
DD34E TEs from cyanobacteria and fungi (T31220, Nostdoc, and Ant1 in Fig 2.3),
although the bootstrap value in the MP analysis was below 50%. Both gambol elements
and these DD34E transposons are larger than Tc1, which usually range between 1.3 and
2.4 kb in size (Table 2.1). No gambol-like elements were found so far in any other
animal species during BLAST searches using the gambol transposase sequences as
queries.
2.4.5 Differences between gambol and Tc1 TIR consensus sequences
As seen in Table 2.1, the TIR lengths within gambol and Tc1 vary widely in An.
gambiae. On average, it appears that the gambol TIRs tend to be longer, the longest of
which is 1096 bp and extends into the ORF of the element. A comparison of the TIR
WebLogo consensus sequences of the gambol and Tc1 TEs from An. gambiae (Fig. 2.4A
and B) shows that they both invariably begin with a ‘CA’ sequence, have a preference for
a ‘G’ in the fifth position, and ‘CA’ in the 11th and 12th position. However, the overall
consensus sequences are significantly different with the gambol elements beginning with
‘CAGGGTTT’, and the Tc1 elements beginning with ‘CANTGNC/T’. This evidence is
in agreement with the distinction between gambol and Tc1. Interestingly, the TIR
consensus of the gambol elements more resembles that of the DD37D transposons (Fig.
2.4C) than that of Tc1.
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2.4.6 Chromosomal densities of the gambol and Tc1 transposons in An. gambiae
Figure 2.5 shows the number of gambol and Tc1 transposons per Mb located on
each chromosomal arm, with the highest and lowest densities located on X and 2R,
respectively. These results are consistent with those reported by Holt, et al. (2002),
which suggested that TE densities within the euchromatic component were highest on the
X chromosome and lowest on the 2R arm. A large proportion of gambol and Tc1 are
located in unmapped scaffolds, which is to be expected if we assume that most of these
scaffolds are largely made up of repetitive DNA. While there is approximately double
the number of Tc1 over gambol TEs, the percentages of the distributions of the two
groups among the chromosomes are approximately the same.
2.5 Discussion
Sequenced genomes are valuable resources for the systematic analysis of the
genomic landscape for divergent TEs and the discovery of novel TEs. Whole genome
investigation offers unique insight into the evolutionary dynamics of TEs and their
contribution to the complex genomic picture we observe today. Our survey of the An.
gambiae genome using a reiterative and exhaustive search program has revealed a rich
diversity of DD34E transposons and uncovered a novel family named gambol (Table
2.1). According to sequence comparison and phylogenetic analysis, gambol elements are
clearly distinct from Tc1 with which they share the same DD34E catalytic triad (Fig. 2.3).
Thus our analysis of the An. gambiae genome not only uncovered a number of new
DD34E transposons, it adds a new family, gambol, to the six defined TE families in the
widely distributed IS630-Tc1-mariner superfamily. No gambol-like elements were found
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thus far in any metazoan species other than An. gambiae. Among all IS630-Tc1-mariner
elements, gambol transposons appear to be more closely related to some DD34E
transposons from cyanobacteria and fungi, which were part of the previously unresolved
DDxE elements (Shao & Tu, 2001). The large size of gambol is also reminiscent of some
of these fungal and archeal DD34E transposons. It is possible that gambol may be the
founding member of a deep lineage of widely distributed DD34E transposons and
additional gambol-like transposons from diverse organisms may be discovered as more
genome sequences become available. It is worth noting that the Tc1 family, the other
DD34E transposon family, has divergent members in vertebrates, invertebrates, and fungi
(Shao & Tu, 2001), indicating a broad distribution.
Despite the current lack of evidence for gambol homologues in other metazoans,
the existence of many gambol elements with divergent transposase sequences in An.
gambiae (up to 62% divergence at the amino acid level) may suggest a relatively long
evolutionary history. Even if we do not include the degenerate elements, gambol clearly
comprises highly divergent members. Such a genomic landscape may result from
multiple invasions (horizontal transfer) by divergent gambol transposons during
evolution. Intra-genomic diversification, which may result from the evasion of host
suppression (Lampe et al., 2001), could also contribute to the divergent composition of
the gambol family. Such host suppression of TE activity may speed up TE
diversification. The above two hypotheses are not mutually exclusive. The observed
intra-genomic diversity is not unique to gambol. An. gambiae Tc1 transposons also
include a highly divergent group of elements (Fig. 2.3). We have also shown that Topi, a
previously identified Tc1 element (Grossman et al., 1999), is comprised of three lineages.
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ITmD37E (DD37E), another family of transposons in the IS630-Tc1-mariner
superfamily, has also only been found in mosquitoes so far. The mosquito-specific
distribution of these unique transposon families is intriguing, although there is no obvious
reason to suggest that mosquito genomes are particularly fertile grounds for unique
families of DNA transposons. In this regard, it is interesting to point out that we have
previously noted an unprecedented diversity in non-LTR retrotransposons in the An.
gambiae genome (Biedler & Tu, 2003).
The gambol transposases are characterized by three common features in their N-
terminal DNA-binding domain: a unique amino acid signature W[I/L/V]DEDC, and two
conserved HTH motifs, namely the PAI and RED domains. At present, the function of
the signature sequence has not been determined. While the primary sequence of the
predicted HTH motifs has undergone numerous amino acid changes, the secondary
structure has been conserved between gambol elements. On the other hand, no changes
appear to have been tolerated in the W[I/L/V]DEDC signature, indicating its functional
importance. The signature is located in the RED domain, which is hypothesized to play a
role in non-specific DNA binding in Tc1 transposons (Vos et al., 1993; Colloms et al.,
1994; Pietrokovski & Henikoff, 1997; Watkins et al., 2004). The PAI domain, on the
other hand, has been shown to mediate the recognition and binding of TIRs in Tc1.
However, the gambol W[I/L/V]DEDC signature sequence is more similar to some
mariner elements (Fig. 2.2), in which the entire N-terminal region is believed to be
important for the efficient and specific binding to TIRs (Auge-Gouillou et al., 2001).
Although it is tempting to suggest that gambol and mariner share similar DNA-binding
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domains, we are not able to confidently infer such a relationship because of the lack of
overall sequence conservation in this region.
Several DD34E TEs may be active as indicated by high sequence identity between
transposon members and the possession of intact TIRs and ORFs. These include Topi,
Tsessebe, Parker, Mango, and AgH1. If active copies can be found, these elements may
provide new transformation tools for the genetic manipulation of mosquito genomes. In
addition, systematic analysis of endogenous transposons in mosquito genomes such as the
study presented here will lead to better-informed design of transposon-based
transformation systems to reduce complication or instability resulting from interactions
between transformation vectors and endogenous TEs (Sundararajan et al., 1999;
Jasinskiene et al., 2000; Atkinson et al., 2001). Efficient transgenic tools will contribute
to the control of mosquito-borne diseases either directly by creating pathogen-refractory
mosquitoes, or indirectly by improving our understanding of mosquito-pathogen
interactions. The existence of a wide array of TEs in the An. gambiae genome is
evidence of their successful spread during certain evolutionary time. Although there are
considerable challenges, it is conceivable that a well-designed transformation system will
be able to help us drive refractory genes into mosquito populations, resulting in reduced
vectorial capacity and the control of mosquito-borne diseases.
2.6 Acknowledgements
TEpost, TEcombine, FromTEpost, TEmask, and TEpipe were implemented by Feng
Zhang and Rui Yang of the Department of Computer Science at Virginia Tech. TEMap
was implemented by Chunhong Mao at Virginia Bioinformatics Institute. We would like
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to thank Jim Biedler for development and assistance with TEpipe, and for many
stimulating conversations. We would also like to thank two anonymous reviewers for
constructive comments and suggestions. This work was supported by NIH grants
AI42121 and AI053203, and the Virginia Experimental Station.
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A number of amino acids not shown
Approximately 60 amino
acids omitted WI/VDEDC
motif A
Ant1
Kiwi IASIREWIDEDCSQSLKELV DAIIFIDE------VGFQVNMRVAYGR ILIMDNVPFHRSID----VRNAIEEGG-HTIML--LPPYSPFFNPIEN-LFSKWK Ozzie VIHIQSWIDEDCSISLKKLK ASIIFIDE------VGFNVSMRTMMGR LLIMDNVAFHKCTA----VREAIIEEG-CEVKY--LPPYSPFLNPIEN-LFSKWK Whistler EEQIRAWIDEDCSISLKKLA YEVIFIDE------VGFNVSMRDTRGR ILILDNVAFHKSYE----VKQKIESYG-YKIMY--LPPYSPFLNPIEN-MFAQWK Mango VESIRAWIDEDCAITLKSLA SGLIYLDE------VGFNVSMRTSKGR YLIMDNVAFHKSQS----VQEAIGTVI-DKPLY--LPPYSPFLNPIEN-MFSKWK AgH1 LEMIKSWVDEDCTTSLKKIS HNFMFVDE------VGFNISLRCKRGW VIFLDNVAFHKTNL----VKQFAEENN-IRLEF--LPPYSPFLNPIEN-MFSKWK Parker VQSIRTWIDEDCTVTLKALA TGIVFLDE------VGFNLSMRTSQGR YLIMDNVAFHKCIE----VKEAIGNEE-DKPLY--LPPYSPFLNPIEN-MFSKWK Pags ISRIRSWIDDDCSISLKKLA AAIIFIDE------VGFNMSMMTMMGR VLIMDNVAFHKCTA----VRETILQEG-CDVKY--LPPYSPFLNPIEN-LFSKWK Lovejoy LVSIH-W-QKYCN---QRKT ANFMFLDE------VGFNVSLRSKRGW IIFMDNVAFHKTNL----VKTFAQNNN-IRLEY--IPPYSPFLNPIEN-MFSKWK Watteau CGAIQNWLADDCGLTLVQLK EKFIFIDE------VGFNVSMRTGYGR VLIMDNVSFHHARA----IKAIVDRYPSFTILF--LPPYSPFLNPIEN-MFSKWK Pi-Pi IDSIKRWIDQDCTISLRKMQ RNIIYIDE------VGMNVSMRATMGR VLVMDNVAFHKCNE----VKECITQHINARLLY--LPPYSPFLNPIEN-MFSKWK Piper ISILQSWIDDDCSISLKKLS DSFIFVDE------VGFNVSLRINRGR IIFMDNVAFHKSRH----VQEFAETNN-IQLQY--IPPYSPFLNPIEN-MFSKWK Julo QNKIRQWLDEDCCLSLKEIK SNMIFIDE------VGFNVSMRLGYGR VLIMDNVRFHHSAR----VEELIETHTGFKIMF--LPPYSPFLNPIEN-MFSKWK M.destructor.mar1 LEA---LLDEDCCQTQEELA SRIITGDE------KWIHYDNSKRKKS IFHHDNARPHVALP----VKNYLENSG-WEVLP--HPPYSPDLAPSDYHLFRSMQ D.mauritiana.mar1 LQA---LLDEDDAQTQKQLA HRIVTGDE------KWIFFVSPKRKKS IFLHDNAPSHTARA----VRDTLETLN-WEVLP--HAAYSPDLAPSDYHLFASMG A.gambiae.ItmD34D.Ag8 IKKIHKMXLNREMK-LIEIA RRNVTMDE------TWLHHYTPESNRQ LFDQDNAPCHKSLR----TMAKIHELG-FELLP--HPPYSPDLASSDFFLFSDLK C.elegans.ITmD37D1 IKKVRGRFRHNSGRSVRAMA RKVLFTDE------KIFCIEQSFNTQN TFQQDGAPAHKHKN----VQAWYESNFPDFIAFNQWPPSSPDLNPMDYSVWSVLE An.gambiae.ITmD37D1 -------------------- HNIIFSDE------KLFTLEETLNKQN CFQQDSPPAHKASI----FQKWCNVLLLFFISASEWPASSPYLNPLDFCIWGYML MsqTc3 KREIVRTAS-NSQKSLKQIK -MMIFSDE-----KK-FNLDGP-DGFN TFQQDNAAIHTSKE----TKQWIKDHKIDLLD---WPARSPDLNPVEN-LWGILV Tc3 ERNVIRAAS-NSCKTARDIR -KVVFSDE-----KK-FNLDGP-DGCR RFQQDNATIHVSNS----TRDYFKLKKINLLD---WPARSPDLNPIEN-LWGILV Sunny KRRIIRATS-NSTKGCLRIK -DVVFNDE-----KKKFKVDGP-NHYS IFQQDNASVQVSKK----TKDYLESMQINTMT---WSAVSSDX--IEN-VWGILL Topi.1 DARIVEMIRADPFKTCTRIK SKIIFSDE------SRINLDGS-DGIK IFQHDNDSKHTSRT----VKCYLANQDVQVLP---WPALSPDLNPIEN-LWSTLK Topi.2 DAQMVEIIRADPFKTCNRIK SKIIFSDE------SRINLDGS-DGTK IFQHDNDSKHTSRL----VKCFLANEDVQVLP---WPALSPDLNPIEN-LWSTLK Topi.3 DAQIVEKVRADPFTTCTRIK MKIIFSDEFSSVQSSPINLDGS-DGIK IFQHDNDSKHTSRT----VRCYLANQDVQVLP---WPALSPDLNPIEN-LWPILK Tsessebe DRKIVNISKKHPFSSAPEIR RRVLWSDE------SKFNRQGS-DGRR MFMQDNDSKHTSGT----VQTWLADNNVKTMK---WPALSPDLNPIEN-LWAIFK Tiang DARMTRLCKADPFKSVRAIR RNVLWSDE------SKVNLVGS-DGKR QFMHDNDPKHTAKA----VKKWFVDQKIDVMN---WPAQSLDLNPIEN-LWKIVK Frisky DRNIAKLAKKNPFTTSKKIK RNILWTDE------SKIVLFGT-KGRR VFQQDNDPKHTSKR----AKSWFIANNIDVME---WPAQSPDLNPIEH-LWKDIK Tc1 DRNILRSAREDPHRTATDIQ AKHIWSDE------SKFNLFGS-DGNS VFQQDNDPKHTSLH----VRSWFQRRHVHLLD---WPSQSPDLNPIEH-LWEELE Tango DTRIVREVKKNPKVTVLEIK KTVLWTDE------SKFELFNR-KRRS VFQQDNDPKHTAKK----TKTFFNSCRIKPLE---WPPQSPDLNPIEN-LWAILD Quetzal RRAIKRLVDAEPEISAQSVA KKVLFTDE------SKFNIFGW-DGTI WFQQDNDPKHTAFN----SRLFLLYNTPHQLK---SPPQSPDLNPIEH-AWELLE S DRLIMRKAIANPRISVRSLA DDVIFCDE------TKMMLFYN-DGPS KFYQDNDPKHKEYN----VRNWLLYNCGKVID---TPPQSPDLNPIEN-LWAYLK Minos KRQLAKIVKADRRQSLRNLA DTIIFSDE------AKFDVSVG-DTRK TFQQDGASSHTAKR----TKNWLQYNQMEVLD---WPSNSPDLSPIEN-IWWLMK Ae.atropalpus.ITmD37E1 RGKVIKAIKRNPNLSDRDLA DGCILMDE------TYVKAEFGQIPGQ MLWPDLASCHYSKT----VIEWYATNGVSVIPKDLNPPNCPQFRPIEK-YWAITK An.gambiae.ITmD37E1 RSKILKTIKGNPNLSDRDLA DGCLLMDE------TYVKADFGQIPGQ MFWPDLASCHYSKV----VREWYAEKGVLFVPKNLNPPNCPQFRPIEK-YWAIMK O.sativa.ITmD39D1 RKKVEIDLSVIAAIPLHQRS ENIIHIDE------KWFNASKKEKTFY WIQQDNARTHLTIDDAQFGVAVAQTGLDIRLVN--QPPNSPDMNCLDLGFFASL- Soymar1 RKRVEIDLSQLREIPLSQRT YNIIHIDE------KWFYMTKKSERYY FIQQDNARTHINPDDPEFVQAATQDGFDIRLMC--QPPNSPDFNVLDLGFFSAIQ T31220 SADILALWEARKDISLEELR ERLVFIDE------TWTATNMTRSHGR VVIMDNLSSHKRPA----VRDRIEAAG-ATLRF--LPPYSPDFNPIEK-AFSRLK Nostoc MKILEEIVEAKNDLTLSEIR ENLVFLDE------AGANLSLIRHSAR CVIMDNCSIHKGGD----IEKLIESAG-AKLIY--LPPYSPDFSPIEN-CWSKIK
VKALCDHLLEKPYLYLDEMA RHLLFVDE------SGCDRRIGFRRTG VIVMDNASFHHSEK----IEELCSQAG-VKIVY--LPPYSPDLNPIEE-FFSELK
Figure continued… 35
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B
Figure 2.1A. Select sections of the multiple sequence alignment of the An. gambiae DD34E TEs and IS630-Tc1-mariner superfamily representatives.
Underlined text above the alignment is the conserved N-terminal amino acid signature of the gambol elements, W[I/L/V]DEDC. Residues of the
catalytic triad are marked with arrows. Italicized TE names indicate those elements which are probably inactive due to various mutations in their
ORFs. 2.1B. Consensus sequence of the gambol W[I/L/V]DEDC signature presented in WebLogo format (http://weblogo.berkeley.edu/logo.cgi),
which shows the frequency and degree of sequence conservation of the amino acids at each position. The most frequent amino acid is on top of the
stack, and the height of each letter is proportional to its frequency. The total height of each stack represents the overall conservation at that position.
Positions without letters means that there is no consensus at that particular position.
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TE N-Terminal Domain (PAI)
SleepingBeauty MGK-SKEISQD-LRKKIV-DLHK---SGSSLGAISKRLKVPRSSVQTIVRKYKHHGTTQSY Tc3 MPRGSALSDTERAQLDVM-KLLN-----VSLHEMSRKISRSRHCIRVYLKDPVSYGT---- Kiwi YEMERGAIKKITSNEDRE-RIVRAYDQGKNMKEIAEMLHLRHSTVHGIVKKYRETGVIE-- Ozzie TSRLN-RKHKTTSNEDRE-RIIAANENGYSTTLIAEMLSINRSTVYSILKKYWKTGEIE-- Whistler QEEHQKRTYRTTTKADRE-RVIAAHENGNRVGAIARMLNMKRQTVAGIIKKFNETSVIE-- Mango TSAATRRVVKTTSDSDRK-RVITAYENGSAPSAISQMLNIKRPTVYGIINKYNATWQIA-- AgH1 -----MPLRKKISLEDKQ-KIVKLYHEEYRMTDIARIMDLNIQTVSRIVHNFLKEGEITNH Parker LDATPRRRNKTTSDEDRK-RVITAYENGVAGKDIALMLNLHRATVYSIIKKFQKTWNVE-- Pags TAPQNRRKNNTTSKADRE-RILAAHENGADTTMISKTLGIKRGTVYAILKKYFKTGDIE-- M.destructor.mar1 MENFENWRKRRHLREVLLGHFFAKKTAAESHRLLVEVYGEHALAKTQCFEWFQRFKSGDFD D.mauritiana.mar1 MSSFV--PNKEQTRTVLIFCFHLKKTAAESHRMLVEAFGEQVPTVKKCERWFQRFKSGDFD
Linker C-Terminal Domain (RED)
SleepingBeauty Y-RSGRR--RVLSPRDERTLVRKVQINPRTTAKDLVKMLEETG-TKVSISTVKRVLYRHNLKG Tc3 --SKRAPRRKALSVRDERNVIRAAS-NSCKTARDIRNELQLS----ASKRTILNVIKRSGVIV Kiwi ATKRTTPNTKKLSQREIASIREWIDEDCSQSLKELVAKLREHHQVVVSTTTVARAIKGFHYSF Ozzie AQRRGGVKQKKLTNAAVIHIQSWIDEDCSISLKKLKSKVLERHGIEVSTSTIARAIKGFNYSF Whistler AGLRGGTRAKKLSTEQEEQIRAWIDEDCSISLKKLAAKVHEAFQITVSKTTIAKVIEGFNYTL Mango AAKRGGTCKKKLSQDAVESIRAWIDEDCAITLKSLAQKVFERHGVHVSISTIAREVKGFNYSF AgH1 SSQKGGPHNKKLTAEQLEMIKSWVDEDCTTSLKKISEKCAREFGVNVCISTIRNYLSEFHYTL Parker AAKRGGNRAKLLPEEAVQSIRTWIDEDCTVTLKALAEKVHERYSVRVSTSTIARQIKGFNYTF Pags AYQRGGAKPRKLTEEAISRIRSWIDDDCSISLKKLADRVWDVYQIRVSTTTISRAVQSFHYSW M.destructor.mar1 TEDKERP--GQPKKFEDEELEALLDEDCCQTQEELAKSLGVT------QQAISKRLKAAGYIQ D.mauritiana.mar1 VDDKEHG--KPPKRYEDAELQALLDEDDAQTQKQLAEQLEVS------QQAVSNRLREMGKIQ
Figure 2.2. The DNA binding domains of Tc3 and Sleeping Beauty (SB) compared to the
predicted HTH motifs of the gambol elements and two mariner TEs with which the gambol
elements share a similar N-terminal signature (XXDEDC). In grey shading, SB: residues that when
changed to alanine destroy transposase/TIR binding (Yant et al., 2004); Tc3: residues that contact
the TIR in a crystal structure (Watkins et al., 2004). Underlined are residues predicted to form
helix structures according to PROF predictions (http://www.predictprotein.org).
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Figure 2.3. Phylogenetic relationships between An. gambiae DD34E and representative IS630-Tc1-mariner superfamily transposases. Shown is an
unrooted neighbor-joining (NJ) phylogram. Maximum parsimony (MP) produced a single tree with similar overall topology. Small numbers are the
percent of the time that branches were grouped together at a particular node out of 2000 bootstrap replications for NJ and MP, respectively. Values
below 50% are not shown. The alignment used to generate trees is the same as shown in Fig. 2.1. All phylogenetic analyses were carried out using
PAUP* 4.0 b10 (Swofford, 2003). New TEs discovered in this study are in larger font. Ag in brackets refers to the Tc1 elements found in An.
gambiae.
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A.
B.
C.
Figure 2.4. TIR consensus sequences in WebLogo format for the first 24 nucleotides of the
gambol (A) and Tc1 (B) TEs from An. gambiae, and ITmDD37D (C) TEs from Caenorhabditis
elegans, C. briggsae, and An. gambiae (Shao & Tu, 2001; Shao & Tu, unpublished). The TIRs
of gambol and Tc1 are listed in Table 2.1.
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0
2
4
6
8
10
12
X 2L 2R 3L 3R UNK
Chromosome
#TEs
/Mb
N Total
gambolTc1
Figure 2.5. Density of gambol and Tc1 TEs per chromosomal arm of the An. gambiae genome.
For both families of TEs, the highest density is on chromosome X whereas the lowest is on
chromosomal arm 2R. UNKN refers to TE copies located in unmapped scaffolds.
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TABLE 2.1. The DD34E transposable elements of Anophles gambiae.
Transposon Molecular Characteristics Copy Number Position of Representative TE TIR Non-autonomous
TE Family Name
Length (bp)
Length (bp) TSD
ORF (aa) First 24bp of 5' TIR "Autonomous"
With TIRs
Without TIRsa Total Accession # Start End
Tc1 Topi.1b 24 1413 TA 332 CACTGGTGGACATTAAAATAGGAA 12 15 73(1) 100 AAAB01008944 5222445 5223857
Tc1 Topi.2b 24 1556 TA 332 CACCGCTGGACATAAAAATAGGAA 10 43 61(1) 105 AAAB01008849 2027739 2029294
Tc1 Topi.3b 24 1446 TA 338 CACCGGTGGACAAAAAGATAGGAA 1 5 11 17 AAAB01008975 147381 148826
Tc1 Tango 224 1670 TA 338 CAGTGGCCGGCAAAATAAAGTGGC 1 26 11 38 AAAB01008960 16265453 16267122
Tc1 Tiangbc 260 2003 TA 333 CAGTGTCGGACAAATCGATAGGAC 6 5 33 (1) 44 AAAB01008987 7538596 7540598
Tc1 Tsessebeb 120 1983 TA 330 CAGTATCGGACATTAAGATAGAAC 10 143 112 (3) 265 AAAB01008968 2949062 2951044
Tc1 Friskyd 25 1690 TA 331 CAATTGTGTTCATTAAAATAGCAG 1 1 9 11 AAAB01008880 3259980 3261669
Tc1 Sunnyeh 65 2547 TA N/A CACTGCCATTAATAATGATAGTCG 0 1 14 15 AAAB01008811 1156482 1159028
gambol Kiwi 335 1880 TA 343 CACCTGTTTCCATCTACGTTCGAA 1 36 7 44 AAAB01008846 12859 14738
gambol Ozzief 680 5956 TA 374 CAGGGTTTACCAAGTCACTTTGCG 1 15 1 17 AAAB01008815 407785 413740
gambol Whistler 229 2453 TA 343 CAGGGTTTACCTAGCCAATCGGGC 1 33 6 40 AAAB01008849 597731 600183
gambol Mango 267 3050 TA 359 CAGGGTTTTCCAGTGGTTCTGATA 1 0 6 (1) 7 AAAB01008879 621801 624850
gambol AgH1g 343 3680 TA 337 CAGGGTTTCGAATCATATAAGGGA 2 24 9 35 AAAB01008960 15668672 15672351
gambol Parker 1096 3660 TA 343 CAGGGTTTTTCAATTGAGTTTTGA 1 22 1 24 AAAB01008960 17976068 17979727
gambol Pags 136 3197 TA 348 CAGGGTTTTACAAGTCAGAATCGA 1 0 2 3 AAAB01005441 35464 38660
gambol Lovejoyh 39 3652 TA 236 CATCGTTCATAAAAAATGCCAAGA 1 0 43 44 AAAB01008948 247393 251044
gambol Watteauhj ND ND ND 339 ND 0 ND 1 (1) 1 AAAB01008906 20898 21914
gambol Pi-Pihj 141 NDi TA 354 CAGGGTTTACCAGCATAATAAACA 0 36 11 (1) 47 AAAB01006919 1 1615
gambol Piperh 483 2989 TA 339 CAGGGTTTTCAACGGTTATATTGA 0 2 12 (1) 14 AAAB01008831 161856 164875
gambol Julohj 217 2533 TA N/A CAGCCGTTGCCGTCAAATTTTGGC 0 17 21 38 AAAB01008815 22575 25107
aThe numbers in parentheses under the "Without TIRs" category are the number of TEs with intact ORFs within this category.
bTopi, Tsessebe, and Tiang were identified by Grossman, et al., 1999.
cTiang (Grossman, et al., 1999) and Crusoe (Hill et al., 2001) are truncated and full copies of the same transposon.
dWarren, et al. identified Frisky and reported the sequence under accession #AF298053.
eSunny is interrupted by two repetitive elements, one 5' and one 3' of the ORF, increasing the representative TE length by 765bp.
fOzzie is interrupted by another TE of approximately 1200 bp 3’ of the ORF.
gAgH1 was identified and characterized by Seok, Shao and Tu, unpublished.
hThese TEs are probably no longer functional, see text for details.
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iThe total length of Pi-Pi cannot be determined because the element is located in a short scaffold which cuts off its 3' end. The TIR length of this element was determined by
alignment of non-autonomous transposon members.
jWatteau, Pi-Pi, and Julo have DD35E motifs.
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Chapter 3
Genomic and evolutionary analyses of Tango transposons in Aedes
aegypti, Anopheles gambiae, and other mosquito species
Permission to reprint granted by Blackwell Publishing Company
Insect Molecular Biology
[Epub ahead of Print - May 2007]
3.1 Abstract
Tango is a transposon of the Tc1 family and was originally discovered in the
African malaria mosquito, Anopheles gambiae. Here we report a systematic analysis of
the genome sequence of the yellow fever mosquito, Aedes aegypti, which uncovered
three distinct Tango transposons. We name the only An. gambiae Tango transposon
AgTango1 and the three Ae. aegypti Tango elements AeTango1-3. Like AgTango1,
AeTango1 and AeTango2 elements each have members that retain characteristics of
autonomous elements such as intact open reading frames and terminal inverted repeats
(TIRs). AeTango3 is a degenerate transposon with no full-length members. All full-length
Tango transposons contain sub-terminal direct repeats within their TIRs. AgTango1 and
AeTango1-3 form a single clade among other Tc1 transposons. Within this clade
AgTango1 and AeTango1 are closely related and they share approximately 80% identity
at the amino acid level, which exceeds the level of similarity of the majority of host genes
in the two species. A survey of Tango in other mosquito species was carried out using
degenerate PCR. Tango was isolated and sequenced in all members of the An. gambiae
species complex, Ae. albopictus, and Ochlerotatus atropalpus. Oc. atropalpus contains a
rich diversity of Tango elements while Tango elements in Ae. albopictus and the An.
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gambiae species complex all belong to Tango1. No Tango was detected in Culex pipiens
quinquefasciatus, An. stephensi, An. dirus, An. farauti, or An. albimanus using degenerate
PCR. Bioinformatic searches of the Cx. p. quiquefasciatus (~10 x coverage) and An.
stephensi (0.33 x coverage) databases also failed to uncover any Tango element.
Although other evolutionary scenarios can not be ruled out, there are indications that
Tango1 underwent horizontal transfer between divergent mosquito species.
3.2 Introduction
Transposable elements (TEs) are mobile genetic units capable of replicating and
spreading in a genome. In some cases, they are capable of escaping their current host
genome to invade a naïve genome in a process referred to as horizontal transfer. TEs are
inarguably dynamic components of their host genomes, and in many organisms, make up
a large proportion of a genome. In the case of Anopheles gambiae, for example, it is
estimated that TEs make up 16% of the euchromatic and 60% of the heterchromatic
regions of the genome (Holt et al., 2002). The interactions between TEs and their host
genomes are complex.
Tc1 TEs are DNA transposons that belong to the IS630-Tc1-mariner superfamily,
a wide-ranging group of transposons found in practically all forms of life, including
bacteria, fungi, plants and animals (Shao & Tu, 2001). These transposons contain a single
gene encoding a transposase, flanked by terminal inverted repeats (TIRs) that define their
5’ and 3’ boundaries (Plasterk et al., 1999). They target ‘TA’ sequences for integration
into the host genome, resulting in ‘TA’ target-site duplications (TSDs) flanking the
integrated transposon (van Luenen et al., 1994). Tc1 elements contain a conserved
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DD34E triad within their catalytic domain, where D is aspartic acid, E is glutamic acid
and ‘34’ represents the number of amino acids between the second and third residue of
the triad. In comparison, mariner elements, another founding member of the IS630-Tc1-
mariner superfamily, contain a DD34D triad within their catalytic domain. This triad has
been shown to be necessary for transposition (Lohe et al., 1997).
Several cases of horizontal transfer of elements in the IS630-Tc1-mariner
superfamily are known, with the best-documented cases for mariner elements (see
Robertson et al., 2002). It is believed that horizontal transfer is an essential step in the
lifecycle of these elements, and that this process has contributed to their widespread
distribution (Robertson & Lampe, 1995). These elements do not require host factors for
transposition, and therefore are not restricted in which genomes they invade. Evidence
suggests that horizontal transfer is the only time upon which selection acts on these
elements (Robertson & Lampe, 1995; Lampe et al., 2003). There are also cases of
horizontal transfer of Tc1 transposons, for example Minos (Arca & Savakis, 2000; de
Almeida & Carareto, 2005) and TCp3.2 (Jehle et al., 1998).
In a recent survey of the DD34E TEs of An. gambiae, we uncovered a Tc1
transposon which we named Tango (Coy & Tu, 2005). There are 38 copies of Tango
within An. gambiae, one of which contains an intact open reading frame (ORF) and TIRs.
Tango is 1670 base pairs (bp) long and contains TIRs of 224 bp. The single ORF codes
for a transposase of 338 residues and contains the typical Tc1 catalytic triad, DD34E.
Here we report a systematic analysis of the genome assembly of the yellow fever
mosquito, Aedes aegypti, which uncovered three distinct Tango transposons, AeTango1-
3. We describe the structural and genomic characteristics of the AeTango elements. We
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determined the evolutionary relationships of Tango elements and found that AgTango1
and AeTango1 are most closely related, sharing approximately 80% amino acid identity.
PCR surveys and database analyses revealed patchy distribution of Tango1 in a number
of mosquito species. We propose that horizontal transfer between divergent mosquito
species best explains the observed species distribution and phylogeny of Tango1
elements.
3.3 Materials and Methods
3.3.1 Identification of Tango elements in the Ae. aegypti genome
The Ae. aegypti genome assembly was downloaded from The Institute for
Genomic Research (TIGR; http://msc.tigr.org/aedes/release.shtml) on October 7, 2005,
which is the version for current annotation. The search strategy for Tango elements
within this genome is similar to the previously described strategy (Biedler & Tu, 2003;
Coy & Tu, 2005), which is a reiterative and exhaustive search based on BLAST (Altschul
et al., 1997) and implemented using a series of computer programs including TEpost,
TEcombine, FromTEpost and TEmask. The strategy is briefly as follows: the full-
length amino acid sequence of AgTango1 was used to search the Ae. aegypti database
during a tBLASTn search with an E-value cutoff of 1e-5. TEpost parsed and organized
the results from this BLAST search and TEcombine removed redundant hits. The output
from TEcombine was used by FromTEpost to generate a non-redundant list of putative
Tango TE nucleotide sequences in fasta format. Representative sequences from this list
were translated to amino acid sequences using the Translate Tool from the ExPASy
webserver (http://www.expasy.ch/tools/dna.html), which were included and used in
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phylogenetic analysis to verify that they are indeed Tango elements. To ensure that we
included all Tango elements, the selection of representatives was liberal at first, which is
why three Tc1 transposons that were thought to be Tango were re-classified as other Tc1
elements after phylogenetic analysis. We used a setting that each transposon is comprised
of copies that are less than 20% divergent from each other. Using the 20% divergence
criteria, the three Ae. aegypti Tango transposons were able to mask all copies of Tango
elements in the genome. The boundaries of Tango elements were determined by aligning
multiple copies of each Tango and by identifying TIRs and the TA TSDs. In sum,
computer programs described above rapidly produced a list of candidate TEs of interest
that were subject to further phylogenetic analysis and manual inspection to verify/clarify
the classification and boundaries of each TE. Computer programs described here were
run on a Dell 530 Linux workstation with twin 2.0 GHz processors, 1.5 Gb RAM, and 80
Gb hard drive. They can be downloaded from
http://jaketu.biochem.vt.edu/dl_software.htm.
3.3.2 DNA Extraction and PCR Amplification
Species analyzed during the PCR survey of Tango include members of the An.
gambiae species complex, (arabiensis, bwambae, gambiae, melas, merus,
quadriannulatus) and four Anopheline representatives outside the complex (albimanus,
dirus, farauti and stephensi). Ae. albopictus, Oc. atropalpus and Cx. p. quiquefasciatus
were also surveyed. Genomic DNA was isolated from individual mosquitoes by
homogenization in 150ul DNAzol/1.5ul polyacryl carrier in 1.5ml tubes following the
manufacturer’s instructions (MRC, Cincinnati, OH). DNA was resolubilized in 50ul 0.1
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X TE. Eight individuals were used from each species, except in the case of An.
bwambae, in which three were used. One microliter of genomic DNA from each
individual was combined into a pool, one microliter of which was subsequently used as
template in degenerate PCR. In the case of An. albimanus, An. dirus, and An. farauti,
genomic DNA obtained from Malaria Research and Reference Reagent Resource Center
(MR4; http://www.mr4.org/) was used. This genomic DNA was isolated and pooled from
an undisclosed number of mosquito individuals by MR4. Hemi-nested PCR was used to
amplify Tango using degenerate primers, which were designed based on the deduced
amino acid sequences of Tango elements from An. gambiae and Ae. aegypti, and span the
conserved C terminal domain of the transposase (Fig 3.2). The primer sequences used for
the first PCR were TangoDegenF1 5’ AARCCNYTNGARTTYTGGAA 3’ (KPLEFWK)
and TangoDegenR1 5’ ABYTTRTTNGTNACNCCNGTYTT 3’ (KTGVTNK and the
first two nucleotides of N/D/Q at the 5’ end of this primer). Reactions were prepared in
an AirClean AC600 Series PCR Workstation (AirClean Systems, Raleigh, NC) to reduce
chances of cross contamination. Negative controls were used in all reactions which
included all reagents for PCR amplification except for template genomic DNA. We also
used degenerate primers for glutamate dehydrogenase as positive control to verify that
the template DNA was of good quality. Touchdown PCR was used to amplify Tango
elements from mosquito genomic DNA, using rTaq polymerase from TaKaRa Bio, Inc.
(Otsu, Japan). Initial denaturation was carried out at 94°C/5 minutes. Subsequent
denaturation and extension were at 94°C/30 seconds and 72°C/30 seconds, respectively.
An initial annealing temperature of 65°C was used. The annealing temperature was
lowered one degree at the end of each cycle until reaching 55°C, at which 14 cycles were
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carried out. All annealing steps were for 30 seconds. One microliter of the PCR reaction
was used in the second-round PCR using TangoDegenF2 5’
AYRYNATGGTNTGGGGNTGYTT 3’ (the last two nucleotides of N, V[I]MVWGC,
and the first two nucleotides of F) and TangoDegenR1 as primers with an annealing
temperature of 55°C and an extension time of 30 seconds.
3.3.3 Cloning and Sequencing of PCR amplified Tango sequences
PCR products were gel purified with Sephadex Band Prep (Amersham
Biosciences, Piscataway, NJ) and cloned into pGEM T-Easy vector (Promega, Madison,
WI), which were then used to transform JM109 bacterial cells (Promega, Madison, WI).
Four colonies were chosen for An. gambiae molecular form M, five colonies for An.
gambiae molecular form S, An. albimanus, An. melas, and An. merus, six colonies for An.
bwambae, Cx. p. quiquefasciatus, and An. quadriannulatus, seven colonies for An.
arabiensis, eight colonies for Oc. atropalpus, and 13 colonies for Ae. albopictus were
chosen and grown up overnight using standard protocols. Plasmids were isolated using
the Wizard Mini Prep kit (Promega, Madison, WI) and sequenced. Automated
sequencing was performed at the Virginia Bioinformatics Institute, Virginia Tech
(Blacksburg, VA). In addition to the above, the PCR product from An. stephensi was
purified directly with GFX PCR DNA and Gel Band Purification kit (Amersham
Biosciences, Piscataway, NJ), and treated as above with 23 clones chosen for sequencing
and analysis. GenBank accession numbers for sequences obtained from degenerate PCR
are (EF423994-EF424048).
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3.3.4 Phylogenetic Analysis
Neighbor-joining, minimum evolution, maximum-parsimony, and/or maximum
likelihood analyses, as implemented by PAUP* 4.0 b10 (Swofford, 2003), were used to
infer phylogenetic relationships between the Tango transposons and representative Tc1
transposase sequences (Fig. 3.2), and for the Tango nucleotide sequences obtained from
degenerate PCR across mosquito species (Fig. 3.4). Bootstrapping was used to determine
confidence of groupings for neighbor-joining, minimum evolution, and maximum
parsimony methods. Modeltest (version 3.7, Posada & Crandall, 1998) was performed on
nucleotide sequence alignment data to determine the best model for maximum likelihood
analysis (Fig 3.4). The number of bootstrap replicates as well as the methods and
parameters used to obtain alignment input files and to generate phylogenetic trees are
described in respective figure legends. Unless otherwise noted, ClustalX version 1.83
(Thompson et al., 1997) was used to generate alignment input files. Minor adjustments to
alignments, when necessary, were made with SeaView (Galtier et al., 1996). Resulting
trees were either viewed directly using PAUP* 4.0 b10 (Swofford, 2003) or using
TreeView (Page, 1996). Amino acid sequence identities (Table 3.1) were converted from
distance data obtained using PAUP* 4.0 b10 (Swofford, 2003).
3.3.5 Analysis of synonymous and non-synonymous substitution rates
SNAP (Synonymous/Non-synonymous Analysis Program)
http://hcv.lanl.gov/content/hcv-db/SNAP/SNAP.html (Korber, 2000) was used to
determine dN, dS and dN/dS ratios for AgTango1 vs. AeTango1, and for a number of
orthologous host genes shared between An. gambiae and Ae. aegypti (Severson et al.,
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2004). Input files for these analyses were generated as follows. Alignments of the amino
acid sequences were made with ClustalX version 1.83 (Thompson et al., 1997). These
alignments were then used to generate the alignment of the corresponding nucleotide
sequences using CodonAlign version 2.0 (Hall, 2004). The resulting alignment was
converted into FASTA format using ClustalX version 1.83 (Thompson et al., 1997) and
used directly as input for the SNAP program using default parameters. The analysis of
Tango1 was performed with representative sequences from each of the two species as
depicted in Table 3.2.
3.3.6 Search for Tango in the Cx. p. quiquefasciatus and An. stephensi databases
The amino acid sequences of AgTango1 and AeTango1-3 were used as query
sequences in a tBLASTN search against the Cx. p. quiquefasciatus trace file, version 3.0.
The database was downloaded on March 15th, 2006 from NCBI
(ftp://ftp.ncbi.nih.gov/pub/TraceDB/) and has approximately 10x coverage with 5 million
sequences and 4.6 billion bases. Also searched was an An. stephensi database generated
from pyrosequencing by our laboratory with 78 Mbp which represents a 0.33x coverage
of the genome. In both cases, a low stringent default e-value cutoff was used which
allowed for matches of e-value of 10.
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3.4 Results
3.4.1 Discovery, genomic and phylogenetic analysis of Tango transposons in Ae.
aegypti
We have previously described that the An. gambiae genome harbors a single
Tango transposon, AgTango1. During our current survey of the Ae. aegypti genome, we
discovered three distinct Tango transposons, namely AeTango1, AeTango2, and
AeTango3. The amino acid sequence identity between AgTango1 and the three Ae.
aegypti Tango transposons ranges from 59.1% to 79.9% (Table 3.1). The 79.9% identity
is found between AgTango1 and AeTango1 (Fig. 3.1A and Table 3.1). The evolutionary
scenarios contributing to the high conservation between Tango1 transposons in two
highly divergent mosquitoes are discussed later. The high level of sequence identity
(>59%) separates the Tango elements from other Tc1 transposons including the three Tc1
elements from Ae. aegypti, Tc1_Ele4-6, that were found during this genome-wide survey
(Table 3.1). As shown in Figure 3.1B, all four Tango transposons align very well in an
alignment that includes other transposons in the Tc1 family. The classification of the
three AeTango transposons was confirmed by phylogenetic analysis showing AgTango1
and AeTango1-3 form a compact clade that is supported by bootstrap (100%, 99% and
62%) during neighbor-joining, minimum evolution, and maximum parsimony analyses
(Fig 3.2). Within the Tango clade, AeTango1 and AgTango1 are closely related while
AeTango2 and AeTango3 are closely related. Tc1_Ele4 is more closely related to the
Tango elements than other Tc1-type elements, as indicated by the high bootstrap values
for the clade comprised of Tc1_Ele4 and all Tango elements (96%, 95%, and 84%, Fig
3.2). However, we decide not to classify Tc1_Ele4 as a Tango element because of the low
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sequence similarity between Tc1_Ele4 and Tango elements (Table 3.1) and the
degenerate nature of the Tc1_Ele4 sequence (Table 3.2).
Like AgTango1, AeTango1 and AeTango2 elements each have members which
retain characteristics of autonomous elements (intact ORF and TIRs), and therefore could
be active (Table 3.2). AeTango3 is a degenerate transposon with no full-length members
and thus its TIRs, TSDs, and length could not be determined. AeTango1 can be further
subdivided into two groups, AeTango1a and AeTango1b. AeTango1b contains elements
that may have undergone an internal recombination event that has lead to the generation
of long TIRs of approximately 780 bp, and a duplication of the ORF such that half of the
ORF is on both the positive and negative strands of the element. Of particular note is that
while AeTango1a is a low-copy number TE, AeTango1b has achieved a comparatively
high copy number of approximately 440 copies. Characteristics of the three other Tc1
transposons discovered during this study are also shown in Table 3.2.
3.4.2 Tango from Ae. aegypti and An. gambiae share structural and molecular
characteristics
AgTango1, AeTango1 and AeTango2 share a number of conserved molecular
characteristics. The overall lengths of the elements, TIRs, and translated ORFs are all
comparable between the transposons (Table 3.2). Additionally, all three elements contain
imperfect subterminal direct repeats (DRs) within their TIRs (Fig 3.3). Subterminal DRs
are also found in the reconstructed and functional DNA transposon Sleeping Beauty, and
have been shown to be the sites to which the Sleeping Beauty transposase binds (Cui et
al., 2002). Also conserved are the nucleotide sequences flanking the subterminal
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terminal repeats on their 5’ ends (Fig 3.3). There is a conserved ‘GG’ dinucleotide
sequence and a conserved ‘TGA’ trinucleotide sequence that is maintained in all three
transposons at the 5’ end of the outer and inner DR, respectively. The reason for this
conservation is not known, however Cui et al. (2002) demonstrated that changes to the
nucleotides surrounding the subterminal DRs alter transposition efficiency. There are
additional DRs within the TIRs of AgTango1 (Fig 3.3). These DRs are perfect, but their
significance is unknown.
3.4.3 Distribution of Tango among mosquito species
We employed a strategy of degenerate PCR followed by sequencing of clones of
PCR products to survey Tango transposons in a number of mosquito species (results
summarized in Table 3.3). The PCR primers were designed according to amino acid
sequences that are conserved between AgTango1, AeTango1, and AeTango2 (Fig. 3.1B).
There is no stretch of amino acid sequence that is conserved between the above three
Tango elements and the degenerate AeTango3. Given the phylogenetic relationship of the
Tango elements shown in Fig. 3.2, our primer design maximizes the chances for an
inclusive amplification of Tango elements. Tango was found in all members of the An.
gambiae species complex by degenerate PCR, but not in the other Anophelines tested,
namely An. stephensi, An. dirus, An. farauti, and An. albimanus (Table 3.3). PCR
products were not obtained for An. dirus and An. farauti using Tango primers. The
integrity of the genomic DNA from these two species was verified using degenerate
primers for the glutamate dehydrogenase gene (data not shown). Twenty-three clones
were sequenced from An. stephensi, a relative of An. gambiae that is in the subgenus
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Cellia, none of which were found to be Tango elements, nor were they from other Tc1
transposons. These results are consistent with a BLAST search of a pyrosequencing
database of An. stephensi (0.33x coverage) in which no Tango elements were found.
Within the Culicine, Tango elements were identified in Ae. albopictus and Ochlerotatus
atropalpus, but not in Culex pipiens quiquefasciatus. A BLAST search of the trace file of
the Cx. p. quiquefasciatus genome (~10x coverage) did not reveal any Tango elements,
consistent with the PCR data. Description of the databases is in the Experimental
Procedures section.
3.4.4 Phylogenetic analysis of Tango sequences in Anopheline and Culicine species
Maximum likelihood, neighbor-joining, minimum evolution, and maximum
parsimony analyses were performed using the nucleotide sequences obtained from the
clones from degenerate PCR, plus AeTango1-3 from Ae. aegypti and AgTango1 from An.
gambiae, rooted with representative Tc1 elements. Figure 3.4 shows the maximum
likelihood tree obtained using a model selected by Modeltest (version 3.7, Posada &
Crandall, 1998). Analyses using neighbor-joining, minimum evolution, and maximum
parsimony algorithms produced essentially the same tree as maximum likelihood
analysis, differing only in the placement of clone sequences within the major groupings.
Bootstrap was performed using neighbor-joining, minimum evolution, and maximum
parsimony algorithms and the bootstrap values are overlaid on the maximum likelihood
tree (Fig 3.4). As shown in Fig. 3.4, two main Tango groups are apparent, one containing
Tango2/Tango3-type elements from Ae. aegypti and Oc. atropalpus, the other containing
Tango1-type elements in Ae. aegypti, Ae. albopictus, Oc. atropalpus, and all species in
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the An. gambiae complex. There are a few interesting points worth mentioning. First, Oc.
atropalpus harbors highly divergent Tango elements of all three types. The fact that we
were able to obtain such a diverse range of Tango in Oc. atropalpus from a sample of
only eight PCR clones suggests a possibility of even greater diversity of Tango in this
species, and testifies to the coverage of our degenerate PCR strategy. All 13 Tango
sequences from Ae. albopictus are Tango1 elements and they form two distinct clades.
All Tango sequences from An. gambiae complex form a monophyletic group, indicating
that only Tango1-type elements are found in species of this complex. The majority of the
Tango sequences isolated from An. gambiae are highly similar to AgTango1, the putative
autonomous element in An. gambiae. Anopheline Tango1 elements are closer to Aedine
Tango1 than to Oc. atropalpus Tango1, which is incongruent to the host phylogeny.
Although the bootstrap values for the Tango1 clade (69%/56%/56%) and the clade
comprised of Aedes and Anopheles Tango1 (93%/79%/67%) are not high, these clades
are supported by all four phylogenetic algorithms including maximum likelihood.
3.4.5 Evolutionary rates of Tango1 and host genes in Ae. aegypti and An. gambiae
The rate of nonsynonymous (dN) and synonymous (dS) changes, and the dN/dS
ratios were determined for the AgTango1 and AeTango1 pair. The same analyses were
performed on orthologous gene pairs described in Severson et al., 2004. dS values for
only four orthologous gene pairs could be determined as the others were found to be
saturated (Fig 3.5). Tango1 had the highest proportion of synonymous changes out of the
genes analyzed. The dN/dS ratio for the Tango1 pair is 0.07, suggesting that Tango1 is
under purifying selection. The selective constraint on Tango1 appears to be similar to that
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of the host gene opsin (dN/dS=0.06) and much higher than vitellogenin gene 1
(dN/dS=0.22) and transferrin (dN/dS=0.29).
3.5 Discussion
The study presented here focuses mainly on a systematic analysis of the Tango
transposons in the yellow fever mosquito, Ae. aegypti and a comparative analysis of the
distribution and sequences of Tango elements in a number of mosquito species. In
addition to providing a systematic view of the diversity, characteristics, genomic
distribution, and abundance of Tango transposons in Ae. aegypti, our analysis points to
possible horizontal transfer of Tango between highly divergent mosquito species and the
possibility of using Tango transposons as genetic tools to genetically manipulate
mosquitoes.
The first clue suggesting possible horizontal transfer of Tango between
mosquitoes is that AgTango1 and AeTango1 are highly similar in sequence, 79.9%
identical at the amino acid level and 70% identical at the nucleotide level. This appears to
be relatively high considering the estimated divergence time between Ae. aegypti and An.
gambiae (145-200 Mya; Krzywinski et al., 2006). To get an idea of how this identity
compared to other peptides shared between these two species, we randomly surveyed 26
known An. gambiae peptides using a tBLASTn search against the Ae. aegypti genome.
The range of amino acid identities was from 28-96% with an average of 43% and a
median value of approximately 61% (Appendix A). The high level of sequence identity
between AeTango1 and AgTango1 may be explained by either a high selection pressure
on Tango1 sequences due to important functions within these genomes or a case of
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horizontal transfer. The analyses of the dN, dS and dN/dS ratios of Tango1 suggest a
relatively high purifying selection pressure, which could result either from purifying
selection acting during horizontal transfer of transposons (Lampe et al., 2003) or from
Tango1 being “domesticated” to serve important functions in these mosquitoes. The
dN/dS ratio of Tango1 falls within the range found for orthologous genes from An.
gambiae and Ae. aegypti. Without values from a large number of orthologous gene pairs,
we are not able to determine whether the evolutionary constraints on Tango1 are
significantly different from those of the host genes. Therefore, sequence identity data and
evolutionary rate analysis by themselves could not distinguish between vertical
transmission and horizontal transfer of Tango1.
Survey of the distribution and phylogenetic analyses of Tango from a wide range
of mosquito species provide support for horizontal transfer of Tango1. Tango1 is present
in Ae. aegypti, Ae. albopictus, Oc. atropalpus, and all species of the An. gambiae species
complex. No Tango was found in the other four Anophelines tested or in Cx. p.
quiquefasciatus using degenerate PCR. However, we can not rule out the presence of
degenerate Tango fragments in these species even with the appropriate negative and
positive controls as described in experimental procedures. On the other hand,
bioinformatic searches of the Cx. p. quiquefasciatus (~10 x coverage) and An. stephensi
(0.33 x coverage) databases also failed to uncover any Tango element, consistent with
our inability to detect Tango in these two species by PCR. Although the inherent
limitation of PCR and genome sequencing projects prevents us from drawing a definitive
conclusion about the absence of Tango in a number of the species surveyed, our current
data are consistent with patchy distribution of at least Tango1, the element for which we
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have the most data. Horizontal transfer of Tango1 is a more parsimonious explanation
than the alternative hypothesis of vertical transmission with multiple losses of Tango1
(once in Cx. p. quiquefasciatus and more than once in the Anopheline lineages). The
multiple losses theory does not explain the high sequence identity between AeTango1 and
AgTango1 either. Moreover, although there are only three main clusters within the
Tango1 clade (An. gambiae complex cluster, Aedes cluster, and Oc. atropalpus Tango1),
there is incongruence between the host phylogeny and the TE phylogeny between these
three clusters (Fig. 3.4). Namely Anopheline Tango1 is more closely related to Aedes
Tango1 than Oc. atropalpus Tango1. Such incongruence is also consistent with
horizontal transfer of Tango1. It should be noted that we are not suggesting a recent
horizontal transfer of Tango1 between Ae. aegypti and An. gambiae. We are proposing
that there has been a horizontal transfer event of Tango1 between the ancestors of the two
species.
Diverse Tango elements (Tango1-3) are found in two Culicine species, Oc.
atropalpus and Ae. aegypti, which is in contrast to the tight cluster of only Tango1 in the
An. gambiae species complex. A logical hypothesis would be that a common ancestor of
the An. gambiae complex is the recipient of the horizontal transfer event. If this is the
case, we would expect that among Anopheline species Tango will be found only in
members of the An. gambiae species complex, and/or its close relatives, depending on
when the horizontal transfer had occurred. It is also possible that the diversity of Tango
elements we observe in the two Culicine species was due to multiple invasions by Tango
in these genomes. Thus we cannot confidently infer directionality of the horizontal
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transfer of Tango1. Expanding the survey of Tango in an even wider range of mosquito
species will shed light on this interesting question.
AeTango1 has achieved a significantly higher copy number in the Ae. aegypti
genome as compared to AgTango1 has in An. gambiae (3.2). The Ae. aegypti genome
(1300 Mb) is approximately 4.5x the size of the An. gambiae genome (278 Mb). In
addition, the organization of single-copy genes relative to repetitive DNA differs between
these two species (reviewed in Rai & Black IV, 1999). Ae. aegypti’s genome is of the
short period interspersion where single copy sequences 1000-2000 bp in length alternate
regularly with short (200-600bp) and moderately long (1000-4000) repetitive sequences.
The organization of An. gambiae is of the long period interspersion where long repeat
(>5600bp) alternates with very long (13,000bp) uninterrupted stretches of unique
sequences. During our initial survey of the Ae. aegypti genome, it appeared that there was
a higher diversity and abundance of Tc1 transposons in Ae. aegypti than Ag. gambiae
(data not shown). It can be speculated that the Ae. aegypti genome is more permissive of
transposon occupation than An. gambiae. Perhaps the TE load in Ae. aegypti has
contributed to the size and organization of Ae. aegypti.
Intact ORFs and the high sequence identity of AgTango1 and AeTango1, along
with the low dN value, may suggest a potentially active or recently active element in
either Ae. aegypti or An. gambiae. If Tango is active, this would represent the first active
TE found in mosquitoes to have potentially undergone horizontal transfer between
divergent mosquito species. This scenario would offer a tantalizing opportunity to study
the biology of these elements in mosquito species. If not currently active, sequence
comparison between the elements may allow for the reconstruction of a functional Tango
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element, as in the case of Sleeping Beauty. Indeed, the ease of gene synthesis and the
existence of well established transposition assay systems make reconstruction a feasible
option. Active Tango may be used as tools to genetically manipulate mosquitoes for basic
research and for control of vector-borne diseases.
3.6 Acknowledgements
An. bwambae samples were a kind and generous gift from Dr. R.K. Butlin and Dr. R.E.
Harbach. We also thank Jim Biedler for stimulating conversations concerning this work
and for reviewing this manuscript. We thank the anonymous reviewers for their
comments. This work was supported by an NIH grant AI 042121 to Z. Tu.
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A AeTango1: MENAVISKIIPLCIRKLVVHDVRNGESHRAVASKYNISKAAVGKILLKQKTFGSVVDRPG 60 MEN +K IPL +RKLVV DV+NGESHRAVA KY+ISK+AVGKI K T GSVVDRPG AgTango1: MENN--TKQIPLAVRKLVVRDVQNGESHRAVAGKYSISKSAVGKIFKKYSTLGSVVDRPG 58 AeTango1 RGRKRKTDARTDAKIMREVKKNPKVTVREIQKTVQLSVSSRTVRRRLVEQGLNSKVARKR 120 RGRKR TD++TD +I+REVKKNPKVTV EI+ T+QL++S RT+RRR++EQG NSK+A+KR AgTango1: RGRKRITDSKTDTRIVREVKKNPKVTVLEIKNTLQLNISDRTIRRRIIEQGYNSKLAKKR 118 AeTango1 PFISKANKAKRLKFAKEHADKPLEFWKTVLWTDESKFELFNQKRRARVWCRSGEELRERH 180 PFISKANK+KRLKFA+EHADKPLEFWKTVLWTDESKFELFN+KRR+ VWC+ GEEL+ER+ AgTango1: PFISKANKSKRLKFAREHADKPLEFWKTVLWTDESKFELFNRKRRSHVWCKPGEELQERN 178 AeTango1 IQGTVKHGGGNVMVWGCFSWGGVGSLVKIDGIMTADSYINILRENLEVSLIQTGLEDKFI 240 IQGTVKHGGGNVMVWGCFSWGG GSLV+I+GIMTAD+YI IL+ENLEVSLI+TGLE+KF+ AgTango1: IQGTVKHGGGNVMVWGCFSWGGAGSLVRINGIMTADTYITILQENLEVSLIKTGLENKFV 238 AeTango1 FQQDNDPKHTAKKTKSFFRSCRIKPLEWPPQSPDLNPIENLWAILDARVDKTGVTNKNNY 300 FQQDNDPKHTAKKTK+FF SCRIKPLEWPPQSPDLNPIENLWAILD RV KTGVTNK+ Y AgTango1: FQQDNDPKHTAKKTKTFFNSCRIKPLEWPPQSPDLNPIENLWAILDDRVKKTGVTNKDKY 298 AeTango1 FEALERAWEELNPQHLQNLVESMPKRLQQVLKAKGGHINY 340 FEALE AWE L+P H++NLVESMPKRLQ VL++KGGHI Y AgTango1: FEALENAWENLDPNHIENLVESMPKRLQLVLRSKGGHIKY 338
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TangoDegenF1 TangoDegenF2 B AgTango1 FAREHADKPLEFWKTVLWTDESKFELFNRKRRSHVWCKPGEELQERNIQGTVKHGGGNVMVWGCFSWGGAGSLVRINGIMTADTYITILQ AeTango1 FAKEHADKPLEFWKTVLWTDESKFELFNQKRRARVWCRSGEELRERHIQGTVKHGGGNVMVWGCFSWGGVGSLVKIDGIMTADSYINILR AeTango2 FARNHISKPLEFWKHVIWSDESKFELFNKKRRLRVWRKSGEGLQDRHLQPTMKHGGGNIMVWGCFSWFGVGNLAQINGIMTAEGYIDILC AeTango3 FARLHMNKFLEFWKRVVWSDEWKFELFNRKRRQRVGRKSDEGLQDKHLQPTMKHGGGNAMVWGCFSWSGVGKLALINGIMTADRYIDILN Tc1_Ele4 FARKYVVKPLEFWKQVLWSDKSKFELFGRKQRARVWTKPGDTLAHKNVQKTLKHGGGNIIVWRCFAWSGIGNLVKMNRAMTADFNISILK Tc1_Ele5 FAKKYINKGSGFWNSILWSDESKFELFGSTKRRRTWQKADERLKDKNICKTVKHGGGSIMLWGCFAANGVGNLDLIDGIMTGASYTSILN Tc1_Ele6 FARKYASCSTDFLKKIVWTDESKFELKNIKKRQTTRCLKSERLQSRFTQPSVKHGGGS-LLVGCFSWKGIGNIVKINKMMTGESYVKILE Quetzal FAKKYVNHPPEFWKKVLFTDESKFNIFGWDGTIKVWRPPGEGLNPKYTAKTVKHNGGGVLVWGCMAANGVGNLQVIDGIMDQYVYINILK Bari.1 FALEYVKKPLDFWFNILWTDESAFQYQGSYSKHFMHLKNNQ--KHLAAQPTNRFGGGTVMFWGCLSYYGFGDLVPIEGTLNQNGYLLILN SB FATAHGDKDRTFWRNVLWSDETKIELFGHNDHRYVWRKKGEACKPKNTIPTVKHGGGSIMLWCGFAAGGTGALHKIDGIMRKENYVDILK Tc1 WAKAHLRWGRQEWAKHIWSDESKFNLFGSDGNSWVRRPVGSRYSPKYQCPTVKHGGGSVMVWGCFTSTSMGPLRRIQSIMDRFQYENIFE Topi FAEEHLAASIFWWSKIIFSDESRINLDGSDGIKYVWRFPNQAYHPKNTIKTLSHGGGHVMVWGCFSWHGTGPLFRINGTLNSEGYRKILS Tc3 FAKNNMGTN---WSKVVFSDEKKFNLDGPDGCRYYWR---DLRKEPMVFSRRNFGGGTVMVWGAFTEKKKLEIQFVSSKMNSTDYQNVLE TangoDegenR1 AgTango1 ENLE--VSLIKTGLENKFVFQQDNDPKHTAKKTKTFFNSCRIKPLEWPPQSPDLNPIENLWAILDDRVKKTGVTN--KDKYFEALENAWE AeTango1 ENLE--VSLIQTGLEDKFIFQQDNDPKHTAKKTKSFFRSCRIKPLEWPPQSPDLNPIENLWAILDARVDKTGVTN--KNNYFEALERAWE AeTango2 ENLE--ESMLKMGLENNNTFPQDNDPKHTAKKTRAVFRSTRIKSMEWPPQSPDLNPIENLWAILDNKVDKTGVTN--KQAYFAALQKAWD AeTango3 ENLE--ESMQKVGEEDNYTFQQDNDHKYTAKKTLAFFRTCRIKPLEWPPQSPDLNPI--LWAILDNKVEITGVNH--KQTHFAVLEKAWD Tc1_Ele4 KKKKNKGLARRIGLEMMFIFQQANDPKHTAFKT-----------------------------SVDK----GDVII--RDKFFEASEKVWT Tc1_Ele5 QNLM--QSVRKLGLGRRFIFQQDNDPKHVCKIANEYFKKKKINVLEWPPQSPDLNPIENLWAILDNRVPLERRTN--KKAFFEEIKAEWA Tc1_Ele6 ENLKP--SLKKMRMT-DYIFQQDNDLKHTSTHAKRYIQSKKFKMFEWPLQSPDLNPIEHLWAILDDKIPIDSRNN--LNNFWKAIQTAWD Quetzal QNLGP--SLEKLGMSQDYWFQQDNDPKHTAFNSRLFLLYNTPHQLKSPPQSPDLNPIEHAWELLERKIRQTRIKN--RVDLENKLKEAWI Bari.1 NHAF--TSGNRLFPTTEWILQQDNAPCHKGRIPTKFLNDLNLAVLPWPPQSPDLNIIENVWAFIKNQRTIDKNRK--REGAIIEIAEIWS SB QHLK--TSVRKLKLGRKWVFQMDNDPKHTSKVVAKWLKDNKVKVLEWPSQSPDLNPIENLWAELKKRVRARRPTN--LTQLHQLCQEEWA Tc1 TTMRP-WALQNVG--RGFVFQQDNDPKHTSLHVRSWFQRRHVHLLDWPSQSPDLNPIEHLWEELERRLGGIRASN--ADAKFNQLENAWK Topi RKMLP-YARQQFGDEEHYIFQHDNDSKHTSRTVKCYLANQDVQVLPWPALSPDLNPIENLWSTLKRQLKNQPARS--ADDLWTRCKFMWE Tc3 LELS---KYLRHYSRKNFRFQQDNATIHVSNSTRDYFKLKKINLLDWPARSPDLNPIENLWGILVRIVYAQNKTYPTVASLKQGILDAWK
Figure 3.1. A. BLASTp alignment showing high sequence similarity between AgTango1 and AeTango1. Expect = e-166; Identities =
270/340 (79%), Positives = 309/340 (90%), Gaps = 2/340 (0%). Alignment was made using NCBI blast server with default settings using
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the bl2seq program (http://www.ncbi.nlm.nih.gov/blast/bl2seq/wblast2.cgi; Tatusova & Madden, 1999). B. Amino acid alignment of the
catalytic domain of the Tango elements from Anopheles gambiae and Aedes aegypti, three additional Tc1 elements from Ae. aegypti, and
representative Tc1 elements from other organisms. ClustalX version 1.83 (Thompson et al., 1997) was used to generate the alignment using
default settings. Degenerate PCR primers to investigate the distribution of Tango in different mosquito species are shaded in grey boxes,
which were designed to maximize amplification of Tango. Conceptual translations of Tango and Ae. aegypti Tc1 nucleotide sequences were
made using ExPASy’s translation tool http://www.expasy.ch/tools/dna.html). Tc1_Ele4 contains an intron, which was removed for this and
all subsequent analyses. SB = Sleeping Beauty. References for Tc1, Tc3, Bari-1, Quetzal, Sleeping Beauty, and Topi are as follows:
Emmons et al., 1983 and Liao et al., 1983; Collins et al., 1989; Caizzi et al., 1993; Ke et al., 1996; Ivics et al., 1997; Grossman et al., 1999,
respectively.
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Figure 3.2. Inferred phylogenetic relationships between Aedes aegypti Tango elements,
AgTango1 from Anopheles gambiae, and representative Tc1 from Ae. aegypti and other
organisms. The amino acid alignment used to generate trees was made with ClustalX version
1.83 (Thompson et al. 1997) using default settings and is the same as shown in Fig. 3.1B, except
that whole transposase sequences were used. All phylogenetic analyses were carried out using
PAUP* 4.0 b10 (Swofford, 2003). Shown is an unrooted neighbor-joining phylogram
constructed using mean character difference as distance measure. Bootstrap information from
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neighbor-joining as well as minimum evolution and maximum parsimony are overlaid on the
neighbor-joining tree. Minimum evolution analysis was performed using the same distance
measure and produced a tree identical in topology to the neighbor-joining tree. Unweighted
maximum parsimony was performed and produced three most parsimonious trees with similar
overall topology to the neighbor-joining tree, differing only in the inter-relationships between
Tc3, Bari-1 and Quetzal. Small numbers are the percent of the time that branches were grouped
together at a particular node out of 2000 bootstrap replications for neighbor-joining, minimum
evolution, maximum parsimony, respectively. Values below 50% are not shown. Bootstrap for
maximum parsimony analysis was conducted using the heuristic search algorithm with 100
random sequence additions per replicate and tree-bisection-and-reconnection branch swapping.
New transposons discovered in this study are in larger font. References for representative Tc1
transposons are the same as those in Fig. 3.1.
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A CAGTGGCCGGCAAAATAAAGTGGCCACTACTTACATTTTTACATTTTTCTAATTTTTTTCGTTAAAAGTGAAGCTAT
CTTTGTGTTATATTTTTTTAAACAATCTTCATTCTTTGCTCTTAAATGTGCAATCAAAATTTATTGAAAAAATCAAA
GGTTTACCAGTACCAAAAATATTTTTAAAAAAATTAGTCCAAATCACACTGACAAAAAAAAAGTGCCCAC
B
Figure 3.3. Conserved features and sequences of the terminal inverted repeats (TIRs) of Tango
transposons. A. The 5’ TIR of AgTango1 from Anopheles gambiae. The TIRs of AgTango1, as
well as the full-length Tango elements from Aedes aegypti, contain subterminal imperfect direct
repeats (DRs), shown in grey shading. Underlined are conserved nucleotide sequences flanking
the DRs shared by all three Tango transposons. AgTango1 also has two sets of DRs as
demarcated with the arrows, not present in the other Tango transposons. B. Weblogo consensus
(http://weblogo.berkeley.edu/logo.cgi) of the TIRs of AgTango1, AeTango1 and AeTango2
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showing the regions of conservation of the nucleotide sequences of these transposons. The
regions with the greatest conservation are those of the subterminal DRs. In both A and B, only
the 5’ TIR is shown.
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Figure 3.4. Inferred phylogenetic relationship of Tango elements. All phylogenetic analyses
were carried out using PAUP* 4.0 b10 (Swofford, 2003). Shown is a maximum likelihood
phylogram rooted with related Tc1 elements. The maximum likelihood analysis was performed
using HKY85+G model and the among-site rate variation shape parameter equals 2.2164. These
parameters were obtained using Modeltest (version 3.7, Posada & Crandall, 1998). Analysis
using neighbor-joining, minimum evolution, and maximum parsimony algorithms produced
essentially the same tree as maximum likelihood analysis. Five hundred bootstrap replicates
were performed using neighbor-joining, minimum evolution, and maximum parsimony
algorithms. The bootstrap values, which are the small numbers at a particular node, are overlaid
on the maximum likelihood tree in the order of neighbor-joining, minimum evolution, and
maximum parsimony. Values below 50% and values for most groupings within the An. gambiae
complex clade were not shown. Uncorrected distance was used for neighbor-joining and
minimum evolution. Bootstrap of maximum parsimony analysis was conducted using the
heuristic search algorithm with 10 random sequence additions per replicate and tree-bisection-
and-reconnection branch swapping. Ten random additions per replicate are sufficient because the
best tree was identified in the first addition in two separate analyses using 100 random additions.
The nucleotide alignment used to generate the trees was based on the corresponding amino acid
alignment in the following manner: Amino acid sequences for AgTango1, Tc1_Ele5 and
Tc1_Ele6 corresponding to the region amplified by degenerate PCR were aligned using ClustalX
version 1.83 (Thompson et al., 1997) using default settings. The alignment was inspected by eye,
and after minor adjustments, was used to align the corresponding nucleotide sequences of these
elements using CodonAlign 2.0 (Hall, 2004). Using this alignment as a guide, the nucleotide
sequence data for the species surveyed using degenerate PCR were added and aligned. This
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resulting alignment was used as input for phylogenetic analyses. AB=Aedes albopictus,
AGM=Anopheles gambiae molecular form M, AGS=An. gambiae molecular form S, AR=An.
arabiensis, AT=Ochlerotatus atropalpus, BW=An. bwambae, ML=An. melas, MR=An. merus,
QD=An. quadriannulatus. Numbers after the species name refer to the clone number.
Sequences have been deposited to GenBank under accession numbers EF423994-EF424048.
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0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
Rack1 Opsin Tango1 Vit TransFe
Num
ber o
f sub
stitu
tions
per
site
dNdS
dN/dS: 0.04 0.06 0.07 0.22 0.29
Figure 3.5. Comparison of selection pressure on Tango1 and four host genes of Anopheles
gambiae and Aedes aegypti. dN and dS values were determined using SNAP
(http://hcv.lanl.gov/content/hcv-db/SNAP/SNAP.html; Korber, 2000). dS=mean number of
substitutions per synonymous site; dN=mean number of substitutions per nonsynonymous site.
Vit=Vitellogennin, TransFe=Transferrin.
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TABLE 3.1. Percent amino acid identities between Tango transposons, three other Tc1 elements from Aedes aegypti,
and representative Tc1 elements Quetzal and Sleeping Beauty.
AeTango1 AeTango2 AeTango3 Tc1_Ele4 Tc1_Ele5 Tc1_Ele6 Quetzala SBb
AgTango1 79.9 61.3 59.1 48.6 47.7 45.9 37.1 38.0
AeTango1 - 62.5 59.1 48.6 47.1 46.9 38.3 38.6
AeTango2 - - 66.9 45.0 45.5 45.6 36.6 36.9
AeTango3 - - - 43.2 44.8 42.3 36.2 35.6
Tc1_Ele4 - - - - 38.1 36.2 33.8 32.9
Tc1_Ele5 - - - - - 45.8 37.8 43.2
Tc1_Ele6 - - - - - - 34.2 32.4
Quetzal - - - - - - - 36.2
aKe et al., 1996
bSB = Sleeping Beauty; Ivics et al., 1997
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TABLE 3.2. The molecular characteristics and copy numbers of Tango transposons from Aedes aegypti and Anopheles gambiae, and three other
Tc1 elements from Ae. aegypti.
Transposable Element Molecular Characteristics Copy
Number Position of Rep TE
Name TIR
Length (bp)
Length (bp) TSD ORF
(aa) First 24bp of 5' TIR Total
(potentially active)
Contig/ Scaffold Start End
AeTango1a 225 1659 TA 340 CACTGATAGGCAAAATAAAGTGCC 7(2) 6525 23825 25483
AeTango1b 779 1589 TA NAa CACTGATAGGCAAAATAAAGTGCC 441 15269 43406 44994
AeTango2 224 1665 TA 336 CAGTGACCGGCACAAAAAAAATCC 25 (1) 5660 81 1745
AeTango3b ND ND ND ND ND 19 22747 6575 7471
AgTango1 224 1670 TA 338 CAGTGGCCGGCAAAATAAAGTGGC 38 (1) AAAB01008960 16265453 16267122
Tc1_Ele4 26 2406c TA ND CAGTACTGGACAAAAAAAAGTACG 4 15410 12848 15253
Tc1_Ele5 21 1660 TA 334 CAGTCAGTGACAAAAGTTAGT 4 11146 68274 69933
Tc1_Ele6b ND ND ND ND ND 4 8087 102991 103926
aMembers of AeTango1b are degenerate and do not have intact ORFs. See text for details.
bNo full length copies of AeTango3 and Tc1_Ele6 were identified, therefore their molecular characteristics could not be determined.
cTc1_Ele4 has two insertions from other transposable elements. One disrupts the 5’ end of the ORF and the other is 3’ of the ORF. Without these insertions, the representative
copy would be 1566 bp in length.
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TABLE 3.3. Distribution of Tango elements among mosquito species surveyed using
degenerate PCR.
Subfamily Species Subgenus PCR Product
Tango/Total Clones
Anophelinae Anopheles arabiensis (AR) Cellia + 6/6
An. bwambae (BW) Cellia + 4/6a
An. gambiae M (AGM) Cellia + 3/4b
An. gambiae S (AGS) Cellia + 5/5
An. melas (ML) Cellia + 4/5b
An. merus (MR) Cellia + 4/5b
An. quadriannulatus (QD) Cellia + 5/6a
An. stephensi (ST) Cellia + 0/23b
An. dirus (DI) Cellia -c N/A
An. farauti (FA) Cellia -c N/A
An. albimanus (AL) Nyssorhynchus + 0/5b
Culicinae Aedes albopictus (AB) Stegomyia + 13/13
Ochlerotatus atropalpus (AT) Ochlerotatus + 8/8
Culex pipiens quiquefasciatus (CX) Culex + 0/6b
aTwo clones from An. bwambae were Quetzal-like (Ke et al., 1996), and one clone from An. quandriannulatus
was a Fot-1-like (Daboussi et al., 1992) transposon
bClones that were not Tango were non-TE sequences
cPCR with degenerate glutamate dehydrogenase primers produced positive results, showing that the genomic
DNA was intact
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CHAPTER 4
Inter-plasmid Transposition Assay to Test Functionality of AgTango Transposase
4.1 Abstract
AgTango is a Tc1 DNA transposable element (TE) from the African malaria mosquito,
Anopheles gambiae. Recently, we discovered several Tango transposons in the distantly
related mosquito species, Aedes aegypti, at high amino acid sequence identity (79.9%).
This observation led us to question whether or not the AgTango transposase was
functional. An endogenous DNA TE from multiple mosquito species that is capable of
transposition in those species would be highly useful for studying TE behaviour, host
interaction, and regulation through comparative analyses. A functional copy of Tango
would enable us to investigate the mechanisms underlying these differences. Therefore,
we sought to test AgTango’s transposase functionality in an inter-plasmid transposition
assay in cell culture. While the positive control for this assay system proved non-
functional, a number of obstacles were overcome in eventual establishment of this
valuable assay system in our laboratory. No transposition recombinants were observed
for AgTango. Remaining problems concerning the system, and future work with AgTango
are discussed.
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4.2 Introduction
In a survey of the DD34E TEs of the African malaria mosquito, Anopheles
gambiae, we identified several potentially active DNA transposons using TEpipe
(Biedler & Tu, 2003; Coy & Tu, 2005). One of these TEs, AgTango, was subsequently
identified through bioinformatics analyses of the distantly related mosquito species,
Aedes aegypti, at high sequence identity (79.9%; Coy & Tu, 2007). Given the estimated
divergence time between these two mosquito species of 145-200 MYA (Krzywinski et
al., 2006), it is unlikely that this TE was transmitted vertically, but rather horizontally,
between the ancestors to the extant species at some distant point in evolutionary time.
Through a degenerative PCR approach, we surveyed a number of mosquito species, and
discovered that An. gambiae and Ae .albopictus have only one Tango transposon, while
Ae. aegypti and Ochlerotatus atropalpus have at several different Tango transposons.
Furthermore, no Tango transposons were identified in Culex pipiens quinquefasciatus or
in Anophelines outside the Anopheles gambiae species complex. These data support the
hypothesis that Tango has been horizontally transferred between mosquito species, and as
such, suggest that Tango may retain the capability of transposition. An element that can
transpose in numerous mosquito species would facilitate studies concerning differential
dynamics of TEs like that observed for Tango.
AgTango is a Tc1 TE that is 1670 basepairs (bp) in length, with an open reading
frame (ORF) coding for a putative 338 amino acid transpose in a single reading frame
(Fig 4.1). It has 224/223bp imperfect terminal inverted repeats with short direct repeats of
approximately 17-18bp within each TIR. This TIR structure, known as indirect
repeat/direct repeat (IR/DR), is found in the reconstructed, active Tc1 TE, Sleeping
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Beauty (Izsvak et al., 1995; Ivics et al., 1996; reviewed in Plasterk et al., 1999). These
direct repeats have been shown to be the cores of the binding sites for the Sleeping
Beauty transposase (Ivics et al., 1997), and unlike the Tc3 element in which the internal
direct repeats are not necessary for transposition (Fischer et al., 1999), elimination of the
internal repeats in Sleeping Beauty obliterates transposition activity (Izsvak et al., 2000).
The N-terminal domain of the Tango transposase has two predicted HTH motifs (Fig.
4.1B and C), termed ‘PAI’ and ‘RED’, which are involved in the binding of the DNA
substrate (Vos et al., 1993; Colloms et al., 1994; Pietrokovski & Henikoff, 1997; Watkins
et al., 2004). The C-terminal domain contains the DD34E catalytic triad which is required
for transposition (Vos et al., 1993; van Luenen et al., 1994; Vos & Plasterk, 1994; Lohe
et al., 1997). Additional features of the putative Tango transposase, common to other Tc1
TEs, include a nuclear localization signal, a GRPR sequence, and glycine-rich box (Fig
4.1b). The GRPR sequence, an AT-hook-like motif, is a conserved feature of Tc1
elements, and is believed to mediate substrate binding in coordination with the ‘PAI’ and
‘RED’ motifs by contacting the DNA in the minor groove of AT base pairs (Izsvak et al.,
2002). The function of the glycine-rich is currently unknown.
In an effort to determine if AgTango was functional, we employed an inter-
plasmid transposition assay carried out in S2 cell culture as described by Arensburger et
al. (2005). This system employs three plasmids, a helper which provides the transposase,
a donor which supplies the substrate, and a target with selectable markers to ‘capture’ and
report transposition events. We obtained the plasmids from Arensburger to serve as a
positive control, and as starting material for the AgTango constructs. Because of the
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ambiguous nature of the pBSHvSacKOα plasmid (Donor Plasmid - described below),
efforts to characterize this plasmid were also undertaken.
4.3 Materials and Methods
4.3.1 Overview of the Inter-plasmid Transposition Assay
To test AgTango’s transposition activity, an in vitro inter-plasmid assay in cell culture
was employed (Sarkar et al., 1997; Arensburger et al., 2005). The system is comprised of
three plasmids: a helper, donor and target. The helper provides the transposase being
tested under the control of an inducible promoter. The donor provides the substrate for
the transposase, namely a cassette with three selectable bacterial markers flanked by the
cognate TIRs of the transposase. This cassette contains a gene for kanamycin (Kan)
resistance, an Escherichia coli origin of replication (ORI), and a coding region for the α-
peptide from the β-galactosidase gene of E. coli (lacZ α). Outside of the cassette resides a
gene for ampicillin (Amp) resistance. The target is a plasmid that is unable to replicate in
E. coli and carries a gene for chloramphenicol (Cam) resistance. These plasmids are
transfected into cell culture, and the transposase is expressed by inducing the promoter.
The transposase, if functional, should recognize the TIRs of the donor cassette and
transpose the entire construct to new locations, one of which being the target. Upon
receipt of the donor cassette, the target plasmid becomes capable of amplification in E.
coli. E. coli carrying the target plasmid then become Kan and Cam resistant. The DNA is
isolated from the cell culture and is electroporated into E. coli, which is then plated onto
Kan/Cam plates. A small amount is also plated onto Amp plates to determine the donor
‘titer’, which is used to calculate transposition rate. Colonies that grow on Kan/Cam
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plates are recombinants between the donor cassette and the target. To distinguish
transposition from other recombination events, the colonies are subcloned and the
plasmids are recovered. A PstI restriction enzyme digest is performed, which produces a
characteristic digest pattern for transposition events. Those plasmids with the correct
digestion pattern are then submitted for sequencing. Transposition events can be further
distinguished by the characteristic ‘TA’ target-site duplications generated through
transposition of IS630-Tc1-mariner TEs.
The plasmids used for making the AgTango constructs were obtained from Dr.
Peter Atkinson (Arensburger et al., 2005), which had been used to test Herves, an active
Class II TE from An. gambiae. In addition to providing starting material for AgTango
constructs, this system also served as a positive control. Dr. Atkinson generously
provided four plasmids, brief descriptions of which are provided below. Detailed
descriptions of their construction can be found in Arensburger et al. (2005) and Sarkar et
al. (1997).
4.3.2 pKhsp70Herves-PEST Helper Plasmid and pKhsp70rp Helper Plasmid
The pKhsp70Herves-PEST helper plasmid (Herves Helper) was constructed from pK19,
and contains the coding region for neomycin phosphotransferase, which confers
resistance to neomycin and kanamycin. The consensus sequence for the three intact
Herves ORFs was used as the coding sequence for the Herves transposase (Arensburger,
et al., 2005). The ORF is flanked by the 5’ and 3’ regulatory regions of a Drosophila
melanogaster hsp70 (Karch et al., 1981; Knipple & Marsella-Herrick, 1988). The hsp70
promoter is strongly upregulated by heat stress and other environmental stressors such as
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heavy metals, and has been demonstrated to drive heterologous gene expression in S2
cells after being subjected to heat-shock. pKhsp70rp helper plasmid (Helper Empty)
duplicates that sequence of the Helper Herves, minus the Herves ORF. Instead, between
the 5’ and 3’ regions of hsp70, a small multiple-cloning site is inserted to facilitate the
cloning of the DNA of interest. Both plasmids were amplified in OneShot Top10 E. coli
cells (Invitrogen Corp., Carlsbad, CA) following manufacturers instructions.
4.3.3 pBSHvSacKOα Herves Donor Plasmid
The pBSHvSacKOα Herves donor plasmid (Herves Donor) was constructed by
amplifying the entire Herves sequence and cloning it into pBluescriptSK+ (Stratagene,
La Jolla, CA). The Herves ORF was then replaced by DNA encoding three genetic
markers: a gene conferring kanamycin resistance, a ColE1 ORI, and the α-peptide coding
region from the β-galactosidase gene of E. coli (lacZ). This produced a Donor Cassette
containing selectable gene markers flanked by the TIRs of Herves. Herves Donor also
contains a gene outside the Donor Cassette, conferring ampicillin resistance to aid in the
propagation of the plasmid, and the determination of donor titer in the transposition assay
(Arensburger et al. 2005, Sarkar et al., 1997). DH5α E. coli cells (Invitrogen Corp.) were
used for amplification of the donor plasmid per manufacturer’s instructions.
The sequence for the Herves Donor as reported by Arensburger et al. (2005)
contained an ambiguous region of approximately 1260bp that started right outside near
the 3’ end of Herves 5’ TIR and continued all the way through the kanamycin resistance
gene (Fig 4.2A). To elucidate the nucleotide sequence in this region, three sequential
rounds of sequencing were performed, the primers for which are listed in Table 4.1.
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Because of the uncertainty of the original sequence, multiple initial primers were
designed for initial sequencing (Table 4.1). Subsequent primers were based on sequence
data obtained from the previous round. To test the functionality of the Donor cassette, the
kanamycin resistance and the lacZ gene, and the ColE1 ORI, the Donor Plasmid was
digested with SpeI, and the fragment containing the 3’ end of Herves 5’ TIR through the
ColE1 ORI (Fig 4.2A) was religated and used in transformation of OneShot Top10 cells
(Invitrogen Corp.). The bacterial were plated onto LB/X-GAL/IPTG/kan (25ug/ml)
plates.
4.3.4 pGDV1 Target Plasmid
pGDV1 is a cloning vector containing an ORI from a plasmid derived from
Corynebacterium xerosis and a chloramphenicol resistance gene (Bron, 1990). It is
replicated at very high copy number in Bacillus subtilis, and is incapable of replicating in
E. coli. Recombination with the Donor Cassette should confer the ability of this plasmid
to be replicated in E. coli. Dr. Atkinson’s laboratory provided plated B. subtilis colonies
on LB plates carrying the pGDV1 plasmid.
4.3.4 Genomic DNA
Genomic DNA was isolated from eight individual An. gambiae mosquitoes by
homogenization in 150ul DNAzol/1.5ul polyacryl carrier in 1.5ml tubes following the
manufacturer’s instructions (MRC; Cincinnati, OH). DNA was resolubilized in 50ul 0.1
X TE. One microlitre from each isolate was combined into a pool which was used as a
template in all PCR reactions except where noted. An. gambiae mosquitoes (G3 strain)
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were obtained from Malaria Research and Reference Reagent Resource Center (MR4;
http://www.mr4.org/).
4.3.5 PCR
PCR was carried out using an Eppendorf Mastercycler Thermocycler 5333 (Eppendorf
Scientific, Inc., Westbury, NY). TaKaRa rTaq DNA polymerase (TaKaRa Bio, Inc.;
Otsu, Japan) was used in all reactions. Originally, Pfu High Fidelity Polymerase
(Stratagene, La Jolla, CA) was used in PCR reactions, but in some cases would not
amplify the PCR product from An. gambiae genomic DNA for an unknown reason(s).
Sephglas BandPrep (Amersham Biosciences Corp.) was used to gel purify PCR products
from agarose gels per manufacturer’s instructions. Except where noted, PCR products
were ligated into pGEM T-Easy vector (Promega Corp.), and ligation products were then
introduced into OneShot Top10 cells (Invitrogen Corp.) following manufacturer’s
instructions. Plasmids were isolated using the Wizard Plus Miniprep DNA Isolation
System (Promega Corp.). Cloned PCR products were sequenced by Virginia
Bioinformatics Institute, Virginia Tech (Blacksburg, VA). All primers used for making
and verifying the Tango constructs are listed in Table 4.2.
A nested PCR strategy was employed for amplifying Tango’s ORF and TIRs in
order to insure amplifying the specific genomic copy of Tango selected for study,
specifically the only copy possessing intact TIRs and ORF. In the first PCR, at least one
of the two primers targeted DNA flanking the Tango element in scaffold number
AAAB01008960. In the second round of PCR, primers were designed that included the
appropriate restriction enzyme recognition sites on their termini to clone the fragments
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into the backbones of Herves plasmids. Templates for the second round of PCR were
25ug of the first PCR product ligated pGEM T-Easy vector. The PCR scheme that was
used for genomic DNA templates was two cycles of 94°C for 2min, annealing
temperature (Tm) for 45s, extension time (ET) at 72°C followed by 29 cycles of 94°C for
30s, (Tm) for 45s, and ET at 72°C with a final extension time of 72°C for 5min. For nested
PCR templates in pGEM T-Easy vector, a PCR scheme of 30 cycles of 94°C for 30s,
(Tm) for 45s, and ET at 72°C with a final extension time of 72°C for 5min was employed.
Tms and ETs (Tms / ETs) are listed below. Standard molecular cloning techniques were
used to create the Tango plasmids as described in (Sambrook et al., 1989) unless
otherwise noted.
4.3.6 Construction of Tango Helper Plasmid
The template for Tango’s ORF was PCR-amplified using TangoTemp F2/R2 primers
(57°C/2min) and ‘TA’ cloned into pGEM T-Easy (Promega Corp.). After sequence
verification, 25ug of the template was used in PCR of Tango’s ORF (58°C/2min). The 5’
primer, TangoORF-F1-XmaI, included the recognition sequence for the XmaI restriction
enzyme and was designed to insure the inclusion of the Kozak sequence ([G/A]NNATGG).
The 3’ primer, TangoORF-R1-Stop-BamHI included a recognition site for BamHI on its
3’ end. Once the sequence of this product was verified, it was cloned into the Helper
Empty plasmid using standard molecular cloning techniques. The construction of the
resulting plasmid, Tango Helper, was verified with primers pKhsp70-F1/R2.
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4.3.7 Construction of the Tango Donor Plasmid
Templates for the 5’ and 3’ ends of Tango were PCR-amplified and subcloned separately,
with primers TangoProTemp-F1/R2 (59°C/1min30s) and Tango3'Temp-F1/R1
(61°C/45s), respectively. After sequence verification, the left end of Tango, including
bases 1–385 plus the flanking ‘GTTA’ from An. gambiae, and the right end, including
1374-1670 plus the flanking ‘TATACA’ on the 3’ end, were PCR-amplified with
Tango5'TIR-F1-KpnI/ Tango5'TIR-R1-XcmI (61°C/30s) and Tango3'TIR-F1-
BamHI/Tango3'TIR-R1-RsrII (62°C/30sec), respectively. The 5’ PCR fragment included
the TIR and all intervening sequence up to the ‘ATG’ start codon of the ORF. The 3’ end
included the last 22 nucleotides of the ORF through to the end of the 3’ TIR. Tango’s 5’
and 3’ ends were transferred into the Donor Plasmid as KpnI and XcmI and BamHI and
RsrII fragments, respectively. This resulted in the removal of all of the Herves’ TIRs
except for a short fragment of approximately 45bp of the 3’ end of the 5’ TIR (Fig. 4.2B).
This was due to the fact that there were no unique RE sites in this region that would allow
for the complete removal of the 5’ TIR of Herves. Tango Donor was verified using the
following primers TangoDonor-F1/F2/F3 and R1/R2.
4.3.8 S2 Cells
Drosophila melanogaster S2 cells (Schneider, 1972) were maintained at 23°C in
Schneider’s Drosophila media supplemented with 10% heat-inactivated FBS, and
0.2U/ml and 100ug/ml of penicillin and streptomycin, respectively (all reagents from
Invitrogen Corp). Initial S2 cultures were obtained from ATCC (Manassas, VA) at
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passage number 517, and all transposition assays were performed in cell passages of 540
or less.
4.3.9 Transposition Assay
Plasmids were purified using the PureYield Plasmid Midiprep System (Promega Corp.).
Prior to the day of transfection, 5x106 S2 cells were plated in each well of a 6 well plate.
The next day, transfection mixes were prepared for each treatment as follows: 7.5ul of
Cellfectin Transfection Reagent (Invitrogen Corp.) was mixed in 100ul of serum free
media (SFM) by inversion and set aside. Helper, donor and target plasmids (2.5, 2.5 and
5.0ug, respectively) were added to 100ul of SFM in a 1.5ml microfuge tube and mixed by
gentle inversion. To control for transfection conditions, one well of cells was transfected
with a plasmid carrying lacZ gene under the control of the baculovirus promoter, OpIE2.
Cellfectin/media mix (107.5ul) was added to each tube, mixed by inversion, and allowed
to sit at room temperature for 20 minutes. Next, 800ul of SFM was added to each tube
and mixed by inversion.
Cells were washed once with 2ml SFM, overlaid with transfection mix, and
allowed to incubate at 23°C for 10 hours. Afterwards, the transfection mix was removed
and replaced with complete media (FBS plus antibiotics – see above). The plate was
sealed with parafilm, and placed in the 23°C incubator overnight. The next day, cells
were placed in pre-warmed oven set at 42°C for 2 hours. Afterwards, they were placed
back in the 23°C incubator overnight. The following day, cells were harvested by
scraping, and DNA was isolated using Promega’s Wizard Genomic DNA Purification Kit
following manufacturer’s instructions. Purified DNA was reconstituted in 30ul of sterile,
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nuclease-free water. Four microlitres of the DNA preparation were used in the
electroporation of 40ul of DH10β E. coli bacterial cells (Invitrogen Corp.) in 2mm
cuvettes, with 2.5kV, 25uF and 200Ω. Cells were allowed to recover in 1ml of SOC
media in a shaking incubator set at 225 rpm and 37°C for 1 hour, after which 5ul was
spread onto LB/X-GAL/IPTG/Amp (100ug/ml) plates. The remainder was spun down
and all but approximately 100ul of the overlying SOC media was removed. Cells were
gently resuspended by vortexing in the remaining SOC media, and spread onto three
LB/X-GAL/IPTG/Kan/Cam plates (15ug/ml and 10ug/ml respectively). The Amp plates,
which were used to determine donor titer, were incubated overnight and the Kan/Cam
plates were incubated for three days. Blue colonies from the Kan/Cam plates were picked
and used to inoculate 3ml of LB broth supplemented with 25ug/ml kanamycin.
Subcultures were used to inoculate LB broth with 100ug/ml ampicillin for counter-
selection. Plasmids were isolated from LB/Kan growth and subjected to PstI restriction
digest. Plasmids with the correct digestion pattern were submitted to VBI for sequencing
with HervesDonor-F1/F2/F3 and R1/R2 (Table 4.3).
Cells transfected with the OpIE2:lacZ construct were assayed for β-galactosidase
activity using Invitrogen’s B-GAL Staining Kit following manufacturer’s instructions.
4.3.10 Bioinformatic Analyses
Helix-turn-helix motifs were predicted with PROF predictions (Rost & Sander,
1993; Rost et al., 1996) at http://www.predictprotein.org. ClustalX for Windows v. 1.83
(Thompson et al., 1997) was used for making alignments. Assembly of the Donor
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Plasmid sequence and maps of plasmids were created with SeqMan Pro and SeqBuilder,
respectively, from the DNA* Lasergene suite, version 7.1.0.
4.4 Results
4.4.1 Analysis of the pBSHvSacKOα Herves Donor Plasmid
The reported sequence of the Donor Plasmid indicated that a PstI restriction digest
should produce three fragments, 622, 894 and 7626bp (Fig. 4.2). This was confirmed
empirically (Fig. 4.3, Lane 2). The 622 and 894bp fragment arise from within the Donor
Cassette, and should be common to all recombinant plasmids. In Arensburger (2005),
only the 622 fragment is mentioned as a diagnostic feature of recombination between the
Donor Cassette and the target plasmid.
Although the name of the plasmid implies that it contains a coding region for
sucrase, no such ORF was found upon the analysis of the supplied sequence. Instead, the
sequence for a large portion of the Hermes TE was found, including both TIRs and part
of the ORF of that element (Fig 4.2A). In addition, the sequence contained an ambiguous
region of approximately 1260bp in size which started immediately outside of the 3’ end
of Herves’ 5’ TIR and continued all the way through the kanamycin resistance gene (Fig
4.2A). A number of preliminary restriction digests produced inconsistent results with the
predicted digest patterns based on the sequence (data not shown). Due to these
ambiguities and inconsistencies, the region that extends from SphI restriction enzyme site
in the middle of the Herves’ 5’ TIR through the PstI site just before the ColE1 origin of
replication (ORI) in the Donor Cassette (Fig 4.2A) was sequenced. The sequencing of the
ambiguous region revealed that it consists mostly of the kanamycin resistance gene (Fig.
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4.2B). Moreover, Hermes was not present in the plasmid as indicated in the supplied
sequence. Lastly, the plasmid is approximately 1.2kbp smaller than reported.
4.4.2 Verification of Tango Constructs
Sequencing of the template for the AgTango ORF revealed two transitions in
nucleotide sequence resulting in two codon changes: GTT ATT and CGA CAA. To
confirm that these changes were not the result of errors in the amplification process,
another PCR reaction was carried out, and the same sequence changes were observed in
the product. The nucleotide differences produce two corresponding changes in the
predicted amino acid sequence of AgTango, V16I and R134Q (Fig. 4.4 and 4.1C).
Because valine isoleucine is a conservative change, and because the site affected is
near the N-terminus of the transposase, the probability that it would affect transposition
activity appears to be small. However, the arginine glutamine change lies between the
NLS and the first ‘D’ of the DD34E catalytic domain. Moreover, arginine is a
significantly larger residue than glutamine, and if protonated, would introduce a positive
charge at a previously neutral position. The corresponding residue in AeTango1 is a
lysine (see Fig. 3.1A), also a positively charged residue. The location and nature of the
substitution suggested a strong probability that catalytic function might be affected,
therefore, efforts to correct this change for future comparisons between the two versions
of the transposase were planned.
The integrity of the Tango Helper Plasmid was confirmed by a double digest with
BamHI and XmaI. The digest produced a fragment slightly larger than 1kb as expected.
The plasmid was sequenced with primers (pKhsp70-F1/R2) designed against the Helper
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Empty backbone facing inwards towards the inserted ORF of AgTango. The data
produced from this sequencing spanned the entire AgTango ORF and the adjacent DNA
of the plasmid, confirming that 1) The entire ORF sequence was present and correct and
2) it was in the correct position and orientation.
In a similar fashion, the integrity and sequence of the Tango Donor Plasmid was
verified by restriction digests and DNA sequencing. To confirm the identity of the 3’ TIR
insert, Tango Donor was digested with BamHI and RsrII restriction enzymes, producing
the fragment of approximately 300bp as expected. Likewise, the 5’ TIR insertion was
verified by digestion with XcmI and KpnI, producing an expected fragment of
approximately 400bp. Sequencing showed that both inserts were correct in sequence and
in orientation. However, sequence data outside of the 5’ end of AgTango’s 3’ TIR
suggested that this region is not as reported.
4.4.3 No Transposition Detected for Herves – No Positive Control
Only general recombinant products were obtained for the Herves system. In three
experiments, 9.9x105 plasmids were screened, out of which 17 recombinants were
recovered (Table 4.4). Any clones passing the ampicillin cross-selection test which had
PstI restriction fragments of approximately 600 and 900bp, regardless of the number or
size of the other fragments, were sequenced. A total of three clones were sequenced, all
of which were determined to be non-transposition recombinants (e.g., Fig 4.5).
Arensburger (2005) observed 12 transposition reactions in a screening of 5.5x105
plasmids. Based on these results, we would expect to have observed transposition
products in our experiments. A number of controls were employed to identify underlying
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problems with this system. In particular, cells were transfected with a plasmid construct
expressing the lacZ gene under the OpIE2 baculovirus promoter, and an ampicillin
resistance gene. This control served two purposes, one to make certain that transfection
conditions were appropriate for the uptake of plasmid DNA into S2 cells, and to verify
our DNA isolation conditions. In cultures transfected with this plasmid, approximately
8% of the cells were stained (26 blue cells/331 total cells). This is within the typical
range of 5-10% observed in Dr. Atkinson’s laboratory (Rob Hice, personal
communication). No staining was observed in non-transfected cells. These observations
indicated that our transfection conditions were appropriate. Furthermore, colonies were
obtained on LB/Amp plates, indicating that the plasmids were reisolated back from the
S2 cells.
4.4.4 No Transposition Events Detected for Tango
An estimated 5.5x105 plasmids were screened in our Tango transposition assay (Table
4.4). Five recombinants were obtained, one of which was ampicillin sensitive and yielded
a passable PstI digest. However, as with Herves, sequencing revealed this was a non-
transposition recombinant.
4.5 Discussion
4.5.1 Potential Problems with the Transposition Assay
The three most critical steps of the assay are 1) transfecting the plasmids into S2 cells, 2)
isolating the plasmids from S2 cells after allowing time for transposition to take place,
and 3) transfecting the plasmids into E. coli to screen for transposition products. We
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know that the initial transfection was successful because 5-10% of the cells transfected
with the OpIE2:lacZ construct exhibited β-galactosidase activity (Rob Hice, personal
communication). Moreover, we were able to isolate Donor Plasmids from the S2 cells
and transfected them into E. coli by electroporation. Because of these observations, it
seems reasonable to assume that these steps are working properly.
Another critical step in the assay is the heat shock treatment. Induction of a heat-
shock response is necessary for the expression of the transposase. No control was
included in the assay for verifying this step. The heat shock response in S2 cells, as in
whole organisms, is temperature and time dependent. It is also affected by whether
heating is gradual or rapid (Lindquist, 1980). Additionally, the optimal temperature and
the magnitude of the heat shock response depend upon the temperature at which the cells
were previously grown (Lindquist, 1980). Heat shock response is typically measured by
one of two methods, by directly monitoring the expression of heat-shock proteins (Hsps)
themselves or by the fusion of the hsp promoter of interest to a reporter gene. By
measuring Hsp expression, Lindquist (1980) determined that the optimal Hsp70 response
in S2 cells for gradual temperature increase (2°C/15 minutes) occurred at 36-37°C for 1
hour. When the cells were rapidly exposed to high temperature, the maximum response
was observed at 37°C with little response at 39°C and above. The implication of this
research on the technical aspects of the transposition assay suggests that, based on the
growth conditions being used to maintain the S2 cells (23°C), and the type of heat-shock
treatment to which they were subjected (rapid), the optimal heat-shock temperature
should be around 37°C for 1-2 hours, not 42°C for 2 hours as noted in Arensburger et al.
(2005). Because heat-shock response can depend on a number of variables, including
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subtle differences between batches of media (summarized in Lindquist 1980), the optimal
heat-shock temperature and time should be determined empirically for our laboratory
through the use of an hsp70:reporter gene construct. This reporter construct should also
be included in future experimentation to control for this critical aspect of the assay.
4.5.2 Donor Plasmid
A number of ambiguities were resolved and corrections made to the working sequence of
the Donor Plasmid, although nothing was discovered that would indicate that there was
anything functionally wrong with the plasmid itself. However, because the transposition
assay did not produce transposition products, sequencing efforts of the plasmid should be
continued in order to confirm its integrity. Having the correct sequence of the Donor
Plasmid is a prerequisite for both successful trouble-shooting and construct design. If
continued use of this plasmid is planned a number of changes should be considered,
including the removal of homologous regions within the Donor Plasmid. This should
reduce chances of homologous recombination and would decrease the size of the
plasmid. In particular, the extra ColE1 ORI outside the cassette should be excised. There
are also several regions outside of the cassette that contain remnants of the lacZ gene that
could be eliminated (Fig. 4.2B). Insertion of a multiple cloning site at the 3’ end of the 5’
TIR would make the system more amenable for exchanging of TIRs. Whether it would be
more efficient to reengineering the existing Donor Plasmid, or to start over from scratch
needs to be evaluated.
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4.5.3 Tango Transposition Assay
Given that the positive control did not yield transposition products, no firm conclusions
can be drawn from the failure to isolate transposition products using AgTango. It is likely
that the problem(s) affecting the positive control are also affecting AgTango’s
transposition assay. However, there are a few points that need to be kept in mind. The
choice of what regions to include, beyond that of Tango’s TIRs, in the donor plasmid
must balance size with the risk that the region beyond the TIR may affect transposition,
either in a stimulatory or inhibitory fashion. Transposition activity of Tc1 TEs has been
shown to drop off exponentially as the size of the TE increases (Fischer et al., 1999;
Izsvak et al., 2000; Karsi et al., 2001). Experimental data from two Tc1 transposons with
the same IR/DR TIR structure, Minos and Sleeping Beauty, indicate that internal
sequences much beyond the TIRs are not required for mobility (Catteruccia et al., 2000;
Klinakis et al., 2000). This is in comparison to PiggyBac and the mariner TE Mos1, for
which internal sequences are necessary for efficient transposition (Pledger et al., 2004; Li
et al., 2005). However, because the regions residing between AgTango’s TIRs and ORF
are short, approximately 200bp total, it was decided to include these regions in the Tango
Donor construct. AgTango’s donor cassette, including the TIRs, is approximately 3.7kb,
which lies within the practical upper limit of 5kb determined for Sleeping Beauty (Izsvak
et al., 2000; Karsi et al., 2001). However, based on previous experimentation with other
Tc1 TEs, a lower rate in transposition efficiency would be expected as compared to the
native size (Fischer et al., 1999; Izsvak et al., 2000; Karsi et al., 2001). If no
transposition products were detected, it may be that the size of the Donor Cassette is
above AgTango’s permissive limit, or that transposition rate is too low for detection.
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Therefore a negative result cannot be interpreted as evidence of Tango’s lack of
functionality, and an alternative means to test activity may be necessary. In particular, the
next step might be to use a system with a smaller substrate (Ivics et al., 1997).
The above brings to point why having a Donor Plasmid more amenable to
exchange of TIRs is highly desirable. It is not known a priori what regions will be
required, enhance, or inhibit transposition – in some studies this may actually be the
focus. The ultimate goal of the investigation is to determine the functional behaviour of
the TE, and elucidating regulatory regions within the element moves towards that goal. A
Donor Plasmid that has short MCS at each TIR terminus would greatly facilitate these
types of studies.
4.5.4 Alternative Methods for Detecting Transposition
The concept behind the inter-plasmid assay is both elegant and simple. This
method simplifies efforts in identifying functional transposases through a rapid screening
process based on selection markers that significantly reduce the number of false
positives, and does not require as many steps for verification of transposition as in other
assays. However, if the problems interfering with the inter-plasmid assay cannot be
identified and resolved, alternative means to test the functionality AgTango’s transposase
should be considered. Lack of results in the positive control not withstanding, no
transposition products from the AgTango transposase in this particular assay system
could be for reasons other than a lack of functionality. In particular, because the activity
of Tc1 transposases drops exponentially as the size of the substrate increases, other means
of testing activity may be more appropriate for these elements. As stated above, the size
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of the Tango Donor Cassette falls within the functional range, but activity may be too
low for detection. Other transposition assay systems with smaller cassettes might be tried
(e.g., Ivics, 1997). Typically, these systems are comprised of two plasmids, one which
provides the transposase and one which provides the substrate, which is usually an
antibiotic resistant gene flanked by the cognate TIRs. These constructs are transfected
into cell culture and transposase expression is induced, as those in the inter-plasmid assay
system. Cells are subjected to the appropriate antibiotic, and those surviving the treatment
are then assayed for chromosomal insertions by Southern blotting. TE display could also
be used in this type of assay. Another means to detect chromosomal insertions is through
the use of TE display, an assay system built upon the concept of RFLP (Casa, et al.,
2000; Biedler, et al., 2003). TE display is an attractive choice because it provides a
genome-wide analysis of insertions, allows for numerous cell passages or cell types to be
analyzed simultaneously, and flanking DNA of the insertions can be determined with
relative ease. Indeed, in comparative analyses of the behaviour of a given transposase
within different cell types as discussed below, TE display would offer many advantages.
4.5.5 Future Directions for Tango
Tango offers a unique opportunity to study TE behaviour in mosquitoes. It resides in
divergent mosquito species’ genomes and appears to have patchy distribution among
mosquito species. Some mosquito genomes have several Tango transposons while others
only have one (Coy & Tu, 2007). This raises a number of questions. For example, are Ae.
aegypti and O. atropalpus are more ‘permissive’ of Tango transposons? Did one Tango
give rise to multiple transposons, or were these genomes invaded several times? Did
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Tango invade An. gambiae and was shortly thereafter inactivated before it could
diversify? Has the An. gambiae genome just not had exposure to other Tango
transposons? The answer to some of these questions probably lies in the host-TE
interaction through host factors and other inhibitory mechanisms. The ideal would be to
identify or develop a functional Tango transposon that could subsequently be used to
investigate the behaviour of the element within these mosquito species to begin to dissect
the mechanisms behind the observed differences of this element. We have an abundance
of resources at our disposal in terms of live mosquitoes and cell cultures from all angles
concerning the Tango transposon to begin elucidating the TE-host interaction within
mosquito. The key to this research is to get the transposition assay working, and to
identify a functional copy of Tango that will work in multiple species of mosquito. Doing
so will lead to a better understanding of the nature of these elements, and possibly leading
to better research and transgenesis tools developed from Tc1 transposable elements.
4.6 Acknowledgements
We are indebted to Dr. Peter Atkinson’s laboratory for the Herves plasmids, and to Rob
Hice for much assistance and advice with the transposition assay. We also thank Jim
Biedler and Ray Miller for stimulating conversations and scientific camradere.
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A.
Tango ORF TIR TIR
D D E
TIR Recognition and Binding Transposition
G-RichNLS
PAI RED
GRPR
D D E
TIR Recognition and Binding Transposition
G-RichNLS
PAI RED
GRPR
B.
* PAI
C.
AgTango1 MENNTKQIPLAVRKLVVRDVQNGESHRAVAGKYSISKSAVGKIFKKYSTLGSVVDRP SB M-GKSKEISQDLRKKIVDLHKSGSSLGAISKRLKVPRSSVQTIVRKYKHHGTTQPSY GRPR RED NLS AgTango1 GRGRKRITDSKTDTRIVREVKKNPKVTVLEIKNTLQLN---ISDRTIRRRIIEQGYN SB RSGRRRVLSPRDERTLVRKVQINPRTTAKDLVKMLEETGTKVSISTVKRVLYRHNLK NLS * AgTango1 SKLAKKRPFISKANKSKRLKFAREHADKPLEFWKTVLWTDESKFELFNRKRRSHVWC SB GRSARKKPLLQNRHKKARLRFATAHGDKDRTFWRNVLWSDETKIELFGHNDHRYVWR Glycine-rich Box AgTango1 KPGEELQERNIQGTVKHGGGNVMVWGCFSWGGAGSLVRINGIMTADTYITILQENLE SB KKGEACKPKNTIPTVKHGGGSIMLWCGFAAGGTGALHKIDGIMRKENYVDILKQHLK AgTango1 VSLIKTGLENKFVFQQDNDPKHTAKKTKTFFNSCRIKPLEWPPQSPDLNPIENLWAI SB TSVRKLKLGRKWVFQMDNDPKHTSKVVAKWLKDNKVKVLEWPSQSPDLNPIENLWAE AgTango1 LDDRVKKTGVTNKDKYFEALENAWENLDPNHIENLVESMPKRLQLVLRSKGGHIKY SB LKKRVRARRPTNLTQLHQLCQEEWAKIHPTYCGKLVEGYPKRLTQVKQFKGNATKY
Figure 4.1. A. Schematic of AgTango, a Tc1 transposable element from Anopheles
gambiae. Direct repeats are demarcated by black triangles in the terminal inverted
repeats. This terminal-inverted repeat structure, known as an inverted repeat-direct
repeat (IR/DR), is also present in the reconstructed and functional Tc1 transposon,
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Sleeping Beauty (Ivics, et al., 1997). B. AgTango’s transposase showing predicted
functional motifs and domains: GRPR, GRPR box; NLS, nuclear localization signal; G-
Rich, glycine-rich box; ‘D’, ‘D’, ‘E’, residues of the catalytic domain. These
characteristics are typical features of a Tc1 transposable element, see text for details. C.
Amino acid sequence alignment of AgTango and Sleeping Beauty, showing the above
motifs and domains within the sequences demarcated by boxes. In grey are the predicted
helix-turn-helix motifs of the paired-like DNA binding domain. In bold are the residues
of the DD34E catalytic triad. Asterisks above the sequence alignment indicate residues
that were found to be different than that predicted by the sequence found within the
Anopheles gambiae genome.
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He rme s
TE
BamHI
ISph
PstI
SphI
IKpn
Sph I
Rsr II
PstI
IXcm
Pst I
1000
2000
3000
40005000
6000
7000
8000
9000
Ambiguous
La
cZ
Herves 3' TIR
ColE1 ORI
Herves 5' TIR
Ampicil lin
Resistance
ColE1 O
RI
9142 bp9142 bp
Donor Plasmid (pBSHvSacKOa)Donor Plasmid (pBSHvSacKOa)
A.
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I
Xcm
BamHI
ISph I
Kpn
Pst I
RsrII
Sph I
Sph I
PstI
PstI
7953 bp7953 bp
1000
2000
3000
4000
5000
6 000
7000
Herves 3' End
AmpicillinRes
i stanc
e
ColE1ORI
Herves 5' End
Kanam
ycinResistance
LacZ
LacZr
ColE1 O
RI
Donor Plasmid - Revised (pBSHvKOa)Donor Plasmid - Revised (pBSHvKOa)
B.
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Figure 4.2. Maps of pBSHvSacOα (Donor Plasmid) and pBSHvOα (Donor Plasmid –
Revised). A. Original map was based on the sequence supplied with the plasmid.
Sections labeled “Ambiguous” and “Hermes” were the two features that were in question
before sequencing. Orientations of features were taken from the map provided with the
plasmid. B. The revised map of the plasmid after incorporating sequence data. The region
that was verified spans from the SphI site in the middle of Herves 5’ End through the PstI
site just outside the ColE1 origin of replication. Other changes include the reversal of the
orientation of the lacZ gene and the reduction in the overall size of the plasmid from 9142
to 7953bp. Small boxes immediately outside the Herves TIRs are partial lacZ coding
sequences.
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8kb 6kb
1kb 750
500
Figure 4.3. A reverse-contrast photograph of an ethidium bromide-stained 1% agarose-
TAE gel showing the results of PstI restriction digest of pBSHvSacOα (Donor Plasmid).
First lane is 1kb marker. Expected fragments based on supplied sequence were 622, 894
and 7626, two of which arise from the Donor Cassette, and will be consistent in all
recombinant plasmids that contain it. No mention of the consistency of the 894bp
fragment was mentioned in Arensburger et al., (2005). The large fragment is estimated
to be 6437bp based upon the revised Donor Plasmid sequence.
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AgTangoAmp MENNTKQIPLAVRKLIVRDVQNGESHRAVAGKYSISKSAVGKIFKKYSTLGSVVDRPGRG AgTango MENNTKQIPLAVRKLVVRDVQNGESHRAVAGKYSISKSAVGKIFKKYSTLGSVVDRPGRG ***************:******************************************** AgTangoAmp RKRITDSKTDTRIVREVKKNPKVTVLEIKNTLQLNISDRTIRRRIIEQGYNSKLAKKRPF AgTango RKRITDSKTDTRIVREVKKNPKVTVLEIKNTLQLNISDRTIRRRIIEQGYNSKLAKKRPF ************************************************************ AgTangoAmp ISKANKSKRLKFAQEHADKPLEFWKTVLWTDESKFELFNRKRRSHVWCKPGEELQERNIQ AgTango ISKANKSKRLKFAREHADKPLEFWKTVLWTDESKFELFNRKRRSHVWCKPGEELQERNIQ *************:********************************************** AgTangoAmp GTVKHGGGNVMVWGCFSWGGAGSLVRINGIMTADTYITILQENLEVSLIKTGLENKFVFQ AgTango GTVKHGGGNVMVWGCFSWGGAGSLVRINGIMTADTYITILQENLEVSLIKTGLENKFVFQ ************************************************************ AgTangoAmp QDNDPKHTAKKTKTFFNSCRIKPLEWPPQSPDLNPIENLWAILDDRVKKTGVTNKDKYFE AgTango QDNDPKHTAKKTKTFFNSCRIKPLEWPPQSPDLNPIENLWAILDDRVKKTGVTNKDKYFE ************************************************************ AgTangoAmp ALENAWENLDPNHIENLVESMPKRLQLVLRSKGGHIKY AgTango ALENAWENLDPNHIENLVESMPKRLQLVLRSKGGHIKY **************************************
Figure 4.4 Amino acid translations of the PCR sequence of AgTango’s ORF
(AgTangoAmp) vs. the sequence from the Anopheles gambiae genome (AgTango). In grey
are the changes in predicted amino acid sequence.
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A. >Herves 5’ 2C1+HervesDonorR2 sequence exported from 1_WellA022C1+HervesDonorR2.ab1 GGACACAGGTGTTTGGGGGTGAAGCATAACAGGATGTTAACAGTCTCTGCAGATACAGGTATAGGAAGCACGAGAGCACCCATCGTCTTTAGCCCCTTCATTCTCCTGCGGCTCTGGTCAGGGAATCTGTAGGCGCAGTCCGCTATTGAGGGAATAAGGAGACTCAGCAATGTGGGTTCTCATCCCATTCTTCTAAAAGTTCCTCCTGCATTTTCAGGGTTATTGTCTCTTGAGCGGATACTTATTGATGTAATAGAAATCATATCAAATAGGGGGGCCGAAACATCACAAGAAAAGTGCCACAAAAGAATAGAGCGTTATTGGGTTGAGAAATTCGCGTTAAGGATCAAGAGTATCAGCTCATTGAACGTGGAATAGGCCGTCATAGGCAGAATCCCTGTTAAATCAGAAAGAATAGACCGAGATAGGGCTGAGTCCCTAATCAGTTTGGAACAAGAGTCCACTATTAAAAGCCGTAAATCGGAACCTCAAAGGGAGAAAAACCGTCTATCAGGGCGAGGGAACGCTGCGGGACGCGTCGAGATAGTCAGGTAAGAAAGGGTAGAGAGCCGGTAAAACGGTAAATCCAAGCGTTAAAGGGAGCCCCCGAGTTAGAGCCACACGGGGAAAGCCGAAGAACGTGGCGCGAAAGGAAGGGCAGAAAGCGAAAGGAGCGGGCATAGGGGGGAAGCAAGATCGCGGTCACCTTGCGCGTAACCACCCAGCTGGCGAAAGGGGGATGCCGGCACAGGGCGCGTCCCGGGTACCATCCAGGTTGTGCCAGTCTTGGGAAGGTAAATCGGCGCGGGCCTAGCGCGCGTAATACCAGCTGGCGAAAGGGGAATTGGGGCAAGGGCCCCCCCTTGGGGTCAATCCGGGATTGACCCGACTTGGTAGCATAAAGAGTCGGCCCTCGAGCGCCAGTAATGTGACTCTCTATCGGTTGATCTGGGTGCCGGGCCGCCCCTCGAGGTCAATTCACTAGTGATTCGATTTTGTAGCAATTAGAGTTGTGCCTCAAGAACCAGAAATGTCCGGTTCAGGCAACTCTTGAAAAGGAGCGAGCGACAGTCGTTCATGTAGTTCATTCCCCCCATTCAAATATGAACGTGAATGGTATGAGCAGGACGTTCACGAAAAGAACGTCCTACCCGTCGAAATGAGAACTCACGGGGTT
B. >Herves 3’ 2C1+HervesDonorF1 sequence exported from 10_WellF012C1+HervesDonorF1.ab1 AAGTTAGCGGATCGCTCGTTCTGTTAATGAAAGAACCGGTAAATTCACGTTCATGGTAATGAATCGAATTGCCCAACCCTAGTAGCAACCGGACCGAATCACTAGTGAATTCGCGGCCCCAGCCGGATCTAATCGCTTTACAAGCTGCCAATCGAAGTTCAATTAGTGAGTTTGAACTGTACTAGTCGTTTTGTTTGAGAGAGAAATGACCTTCTCGTGCTTTGAGGAAGAGAGGAAAATTACACTAATACGTTTATGGTCACTAAATCCATATGATAAATACATAGATCACGTGTGTTCGGTTCGTTGTTGTATAGGTGATTTTGACCGTTATTTTTGAATACGTTCGATTTCTTCAATGTGTTGTAGGCTTATGGTAATAAACATTGCTGAAACCGTGATTGTATTGATAAATTGATCTTTGATATGCCATTTGTTGTGATATTTTGACTTGTAAACCACAAATTGATCTACGCTCCATCGAAATAAGTGAAAATTTAATAATTGCACGGCGATCAAAGGTAACATTAGTCTTGGTAGTTAAAAACAAAAACATAAAGTGTTGTATGAAACATTTCCACAGATATGATGGCTCCAACAAACGCAACAACAAGCCCTGTCTGGGATCATAATCGAATTGACGGTATCGATAAGCTTGATCTAGAGTCTGAGGTCTGCCTCGTGAAGAAGGTGTTGCTGACTCATACCAGGCCTGAATCGCCCCATCATCCAGCCAGAAAGTGAGGGAGCCACGGTTGATGAGAGCTTTGTTGTAGGTGGACCAGTTGGTGATTTTGAACTTTTGCTTTGCCACGGAACGGTCTGCGTTGTCGGGAAGATGCGTGATCTGATCCTTCAACTCAGCAAAAGTTCGATTTATTCAACAAAGCCGCCGTCCCGTCAAGTCAGCGTAATGCTCTGCCAGTGTTACAACCAATTAACCAATTCTGATTAGAAAAACTCATCGAGCATCAAATGAAACTGCATTTATTCATATCAGATTATCATACCATATTTTGAAAGCCGTTCTGTATGAAGGAGAAACTCACGAGGCAGTTCATAGATGCAGATCTGTATCGTCTGCGATCGACTCGTCAACATCAATACACTATATTCCCCTCGTCAAATAGTTATCAGGTGAGAAACACCATGATGACG
Figure 4.5 Sequences from the 5’ (A) and 3’ ends (B) of a Herves recombinant
obtained in the interplasmid transposition assay. These sequences are characteristic of
all recombinants obtained passing the selection process. Terminal inverted repeats are
underlined and in bold. Target site duplications are in grey, and are the same sequences
as those found in the Donor Plasmid. All of the sequence obtained for both ends comes
from the Donor Plasmid except for the region in yellow highlighting, which comes
from the Helper Plasmid. For this particular transposable element, the target site
duplication sequences should be different than those found in the Donor Plasmid, and
the sequence outside of the terminal inverted repeats should be that of the Target
Plasmid.
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Table 4.1. Oligonucleotide primers to sequence the ambiguous region
of pBSHvSacKOα Donor Plasmid.
Oligo Name Sequence 5' to 3' Worked?
First Round pBSHvSacKOα-F1 CTTGATCTAGATAAGCTTCTCG N pBSHvSacKOα-F2 CATTTCCACAGATATGATGG Y pBSHvSacKOα-F3 CACAGATATGATGGCTCCAAC Y pBSHvSacKOα-R1 ATCCAGCCAGAAAGTGAG Y pBSHvSacKOα-R2 TGCTGACTCATACCAGGC Y
Second Round Kan-F1 GCTCATAACACCCCTTGTATTACT Y Kan-F2 TTCTGGCTGGATGATGG N Kan-R1 GCCTCTTCCGACCATC Y Kan-R2 TGATGCGAGTGATTTTGAT Y Third Set Kan-F3 CAGCAACACCTTCTTCACG Y Kan-F4 CAGACCTCAGACCTGCAG Y Kan-F5 ACAGGAAACAGCTATGACCAT N
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Table 4.2. Oligonucleotide primers to create Tango inserts for Tango Helper and Donor plasmids for inter-plasmid transposition assay, and to sequence recombinants to identify transposition reactions.
Oligo Name Sequence 5' to 3' Purpose
Helper TangoTemp-F2 GCATTAGCATTTGGCATTG Create template for Tango ORF TangoTemp-R2 AATTCACTCCTTATCGTTCG Create template for Tango ORF TangoORF-F1-XmaI cccgggAAAATGGAAAACAACACAAAAC† Amplify Tango ORF with XmaI RE site TangoORF-R1-Stop-BamHI ggatccTTAATATTTTATGTGCCCCCC† Amplify Tango ORF with stop codon and BamHI RE site pKhsp70-F1 CTAAGCGAAAGCTAAGCAAAT Verify Tango ORF insert in Helper pKhsp70-R1 AACTTAAGCCAGGAACTGAA Verify Tango ORF insert in Helper
Donor TangoProTemp-F1 AGCGAACAACAGCACAAC Create template for Tango 5' TIR TangoProTemp-R1 ACACCCCCAAACCATAAC Create template for Tango 5' TIR Tango5'TIR-F1-KpnI ggtaccGTTACAGTGGCCGGCAA† Amplify Tango 5' TIR to go into Donor with KpnI RE site Tango5'TIR-R1-XcmI ccatcatatctgtggCATTTTGTGCAACTGAGCC† Amplify Tango 5' TIR to go into Donor with XcmI RE site Tango3'Temp-F1 CAAAGGGGGGCACATAAA Create template for Tango 3' TIR Tango3'Temp-R1 ACTACAAACGAGCACCAACG Create template for Tango 3'TIR Tango3'TIR-F1-BamHI ggatccCAAAGGGGGGCACATAAA† Amplify Tango 3' TIR to go into Donor with BamHI RE site Tango3'TIR-R1-RsrII cggtccgTGTATACAGTGGCCGGCA† Amplify Tango 3' TIR to go into Donor with RsrII RE site TangoSeqDonor-F1 CCAAAGGGGGGCACATAA Verify Tango 3' TIR inserts in Donor TangoSeqDonor-F2 AGGAAGGGAAGAAAGCGAAA Verify Tango 3' TIR inserts in Donor TangoSeqDonor-F3 TTGACATGAGGTGTTTAGGCA Verify Tango 3' TIR inserts in Donor TangoSeqDonor-R1 AGCGAGGAAGCGGAAGA Verify Tango 5' TIR inserts in Donor TangoSeqDonor-R2 TGCCTAAACACCTCATGTCAA Verify Tango 5' TIR inserts in Donor
Tango Transposition Reaction TangoDonor-F1 CCAAAGGGGGGCACATAA Verify 3' end of Tango Donor Cassette in Target TangoDonor-F2 GCCAAACGAATGTCTAGATCGA Verify 3' end of Tango Donor Cassette in Target TangoDonor-F3 GATATCGAGCTCGCTTGGAC Verify 3' end of Tango Donor Cassette in Target TangoDonor-R1 GATCGATTTGCATAGTGGGC Verify 5' end of Tango Donor Cassette in Target TangoDonor-R2 GTTGGAGCCATCATATCTGTGG Verify 5' end of Tango Donor Cassette in Target †Lower case font indicates the restriction enzyme recognition site added to the primer to facilitate molecular cloning.
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Table 4.3. Oligonucleotide primers used to verify Herves transposition events in products of inter-plasmid assay.
Oligo Name Sequence 5' to 3'
HervesDonor-F1 TGTGGTTAGTTGACGTGAACG
HervesDonor-F2 GTGTTAAAATGTGTTGGTTGCC
HervesDonor-R1 GTAGGTCGTTCGTTCTTTAGGATG
HervesDonor-R2 AATGGGGGGAATGAACGAC
HervesDonor-R3 GGCTGAGTGAGAAAGTATGCTGAT
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Table 4.4 Results for inter-plasmid transposition assay for Herves and AgTango. TE
Construct Number of
Experiments Number of Plasmids Screened
Number of Recombinants
(Kan/Cam Resistant)
Number Passing
Amp Cross-Selection
Number Passing
PstI Digest
Number of Plasmids
Sequenced
Number of Transposition
Events
Herves 3 987,000 17 3 3 3 0 AgTango 2 553,000 5 0 1 1 0
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APPENDIX A. Identities at the amino acid levels of different gene products and Tango
between Anopheles gambiae and Aedes aegypti
ENSEMBL Protein Family % AA Identity ENSEMBL ID
Nanos 28 ENSANGP00000020428Immune Response Serine Protease Related ISPR5 (Fragment) 37 ENSANGP00000021259
Putative Apyrase Precursor 50 ENSANGP00000015382
Toll Receptor Precursor 50 ENSANGP00000029576
Transferrin 50 ENSANGP00000021949
Caspase 3 Precursor <53 ENSANGP00000021365
Lectin <53 ENSANGP00000010670
30E5 11 <56 ENSANGP00000022917
Vitellogenin 1 57 ENSANGP00000012892
Gustatory Receptor 66A <58 ENSANGP00000021675
Cytochrome P450 60 ENSANGG00000020445
Chymotrypsin 2 Precursor <61 ENSANGP00000026162
Frizzled Precursor <61 ENSANGP00000019989
PAP 63 ENSANGP00000002978
Serine Protease Inhibitor <64 ENSANGP00000023182
Zinc Carboxypeptidase A Precursor 64 ENSANGT00000021052
Transcription Factor 65 ENSANGP00000018140
Scavenger Receptor Class B Member <67 ENSANGP00000012652
Antennal Carrier AP 2 69 ENSANGP00000027277
25 KDA GTP-Binding Protein (Fragment) 70 ENSANGP00000023777
Beta 1 3 Glucan Binding Precursor BGBP 70 ENSANGP00000016221
Odorant Receptor (GPROR8) 70 ENSANGP00000007927
Tango 80 N/A
White Protein 86 ENSANGP00000007325
Glucose 6 Phosphate 1 Dehydrogenase 89 ENSANGP00000018551
Opsin 90 ENSANGP00000011949
Receptor of Activated Protein Kinase C1 96 ENSANGP00000012560
Identities were obtained by randomly surveying 26 known An. gambiae peptides using a
tBLASTn search against the Aegypti aegypti genome. Values proceeded by a ‘<’ indicates
that the hit did not span the entire query peptide, and that the identity is therefore presumed to
be less than that reported by the BLAST program. In each case, the highest hit was used for
comparison. 129
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MONIQUE R COY
Department of Biochemistry 755 E Main StreetBlacksburg VA 24061 Christiansburg VA 24073(540) 231-1394 (540) [email protected]
EDUCATIONAL PhD Biochemistry June 2007 BACKGROUND Virginia Polytechnic Institute and State University, Blacksburg Virginia Dissertation Title: Analysis of the DD34E transposable elements of the African
malaria mosquito Anopheles gambiae and the identification and characterization of an active transposase
BS Zoology December 1997 Highest Honors University of Florida, Gainesville Florida Thesis Title: Phylogenetic relationships of North American woodpeckers HONORS AND Sigma Xi Phi Beta Kappa AWARDS Gamma Sigma Delta Phi Kappa Phi Keystone Symposia Travel Award 2003 Golden Key Nat’l Honor Society Eheart Travel Award 2003, 2005 Kendall King Award, 2006 Air Force Office of Scientific Research Travel Award 2006 Outstanding Doctoral Student of the Year – Agricultural and Life Sciences 2007 WORK Laboratory Technician 2000-2001 EXPERIENCE Virginia Commonwealth University, Richmond Virginia Determined the ability, time, and dose dependence of EtOH, H2O2, and
deoxycholate to induce apoptosis in AGS and Caco-2 cells as measured by the terminal deoxynucleotidyl transferase dUTP nick-end labeling assay; maintained cell cultures; ordered reagents and supplies
Laboratory Technician 1998-2000 University of Florida, Gainesville Florida Studied the effect of dietary zinc and immunological challenge on the protein
expression levels of zinc transporters, ZnT-1 and ZnT-2, in rats, mice and BHK cell line by western blotting and immunofluorochemistry; designed, developed and purified custom antibodies
TEACHING Graduate Teaching Assistant Spring 2002 EXPERIENCE Concepts in Biochemistry UNIVERSITY Graduate Honor System Panel Member May 2004 – June 2006 SERVICE Graduate Student Mentor Fall 2005 MEETING Fifth International Symposium on Molecular Insect Science 2006ABSTRACTS Tango: A DNA transposable element that dances among mosquito species
American Society of Tropical Medicine and Hygiene 2005 Tango: A DNA transposable element that dances among mosquito species Keystone Symposium 2003 Transposition and Other Genome Rearrangements Five novel families of hAT-like miniature inverted repeat transposable elements
130
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in the Fugu rubripes genome PUBLICATIONS Cui, L., et al. (2000). Prouroguanylin overproduction and localization in the
intestine of zinc-deficient rats. J Nut 130 (11) 2726-2732. Coy, M.R. and Z. Tu. (2005). Gambol and Tc1 are two distinct families of DD34E
transposons: Analysis of the Anopheles gambiae genome expands the diversity of the IS630-Tc1-mariner superfamily. Insect Mol Biol 14 (5) 537-546.
Coy, M.R. and Z. Tu. Tango transposons in Aedes aegypti, Anopheles gambiae, and
other mosquito species: A possible case of horizontal transfer. Insect Mol Biol In press.
Nene, V. et al., 2007. Genome sequence of Aedes aegypti, a major arbovirus
vector. Submitted to Science. M. Coy is one of the four co-authors from the Tu laboratory.
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